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Endocrine Care |
SEMPR, Serviço de Endocrinologia e Metabologia do Hospital de Clínicas da Universidade Federal do Paraná (A.M.R., R.B.R., H.D.L.S., C.L.B.), Curitiba, Brazil; Neurology Division (S.M.D.A.), Department of Internal Medicine, Hospital de Clinicas da Universidade Federal do Paraná, Curitiba, Brazil; and Laboratório Frischmann Aisengart (P.A.N.), Curitiba, Brazil
Address all correspondence and requests for reprints to: Cesar Luiz Boguszewski, M.D., Ph.D., Serviço de Endocrinologia e Metabologia do Hospital de Clínicas da Universidade Federal do Paraná, Rua Padre Camargo, 262, Curitiba, PR, Brazil, CEP: 80060-240. E-mail: . cesarlui{at}hc.ufpr.br
Abstract
The aim of the present study was to evaluate the cerebrospinal fluid (CSF)/serum leptin ratio during pharmacological therapy for obesity with centrally and peripherally acting drugs. Thirty-one obese women (mean age, 32.3 ± 10 yr; body mass index, 38.2 ± 5.2 kg/m2; body fat, 43.3 ± 5.4%) were studied before and 2 months after a weight loss program consisting of a balanced diet (1200 kcal/d) plus drug therapy. The patients were randomly assigned into three study groups: group I, fenproporex 25 mg/d (n = 10); group II, sibutramine 10 mg/d (n = 10); and group III, orlistat 120 mg tid (n = 11). Body fat, measured by dual-energy x-ray absorptiometry, and serum and CSF concentrations of leptin were examined at baseline and 2 months after therapy. At baseline, clinical and biochemical characteristics of the groups were similar. All of the women lost weight, approximately 7.0% of their initial body weight, and the reduction was not different among the groups. Serum leptin fell significantly after 2 months in all groups, and the decline was proportional to the reduction in body fat, because leptin levels adjusted for body fat did not change after treatment. CSF leptin levels showed a significant decrease after 2 months in all groups, and this decline was higher on group III compared with group I (P = 0.006). After therapy, the CSF/serum leptin ratio did not change in group I (1.57 ± 0.3 to 1.72 ± 0.62%) and group II (1.78 ± 1.01 to 1.69 ± 1.27%), whereas it declined significantly in group III (1.65 ± 0.43 to 1.09 ± 0.47%; P < 0.01), corresponding to a decrease of 33.3 ± 22.5% for the CSF/serum leptin ratio. The percentage change in group III was significantly different from the positive variation on group I (11.9 ± 42.1%; P = 0.006) and close to the statistical significance compared with the negative variation seen in group II (-7.6 ± 27.8%; P = 0.06). Our results showed that the CSF/serum leptin ratio decreased after weight loss in obese women treated during 2 months with orlistat, whereas this ratio did not change in this period of time in obese women treated with fenproporex and sibutramine.
LEPTIN IS AN adipocyte-derived hormone with important hypothalamic effects on regulation of food intake and energy expenditure (1, 2). Leptin receptors are highly expressed at choroid plexus and brain endothelial cells, where they possibly mediate leptin transport into the central nervous system (CNS) (3, 4). This leptin transport seems to be performed by a saturable system that is susceptible to physiological and pharmacological manipulation (5, 6, 7). Serum leptin levels are elevated in most overweight individuals; the leptin transport into the brain, as evaluated by the cerebrospinal fluid (CSF)/serum leptin ratio, is reduced in obese subjects, suggesting a resistance to the central actions of leptin in human obesity (8, 9, 10, 11). It is likely that the decreased transport of leptin into the brain in obese subjects results from defects at either the central signal transduction receptor or the blood-brain barrier transporters (7).
Obesity is a chronic, multifactorial, and costly disease whose prevalence is increasing in many countries of the world (12). Pharmaceutical strategies in the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, or fat metabolism (13). Monoamine neurotransmitters, such as noradrenaline, dopamine, and serotonin, have been implicated as effectors in the CNS control of energy homeostasis and are targets for several appetite suppressants used for obesity treatment (14). Very few drugs of this group are approved for clinical use due to their potential for abuse and dependency (15). In Brazil and in some other countries, amphetamine-derived agents such as fenproporex (N-2-cyanoetil-amphetamine) are licensed under restricted control for treating obesity (16). Fenproporex acts by releasing or blocking neuronal reuptake of noradrenaline and dopamine (17, 18). Sibutramine, which is also a ß-phenethylamine derivate, is thought to be devoid of amphetamine-like abuse potential and is available worldwide as an antiobesity drug (13, 19). Sibutramine reduces food intake by blocking reuptake of noradrenaline and serotonin in the brain (13, 14, 19). Another class of antiobesity drugs includes those with actions on fat absorption. Orlistat (tetrahydrolipstatin) is a hydrogenated derivative of a bacterial lipase inhibitor that blocks pancreatic lipase and decreases triglyceride digestion in the intestinal lumen. It is minimally absorbed, thus exhibiting no systemic direct effects in the regulation of body weight (13, 20, 21).
Little is known about the impact of appetite suppressants on the leptin system. In the present study, three groups of obese women were examined before and 2 months after weight loss induced by caloric restriction and medical therapy with fenproporex, sibutramine, and orlistat, to investigate the effects of antiobesity drugs on the CSF/serum leptin ratio, which has been clinically used as an indirect approach to estimate leptin transport into the brain (5, 6, 7, 10, 11, 22, 23, 24, 25).
Subjects and Methods
Subjects and study groups
Approval for the study was obtained from the Ethics Committee of the Federal University Hospital of Parana, and informed consent was obtained from all subjects before their participation. One minor patient was included in the study, and informed consent was obtained from her parents. The study group consisted of 31 ambulatory premenopausal obese women [body mass index (BMI) >30 kg/m2], between 16 and 50 yr of age, who were seeking pharmacological therapy for obesity at the Endocrine Unit of our University Hospital. None of the women presented with or had previously suffered from other serious concomitant diseases. Before the study, each participant underwent a standard physical examination and laboratorial tests to rule out abnormalities. The patients were randomly assigned to receive fenproporex 25 mg/d (group I, n = 10), sibutramine 10 mg/d (group II, n = 10), or orlistat 120 mg tid after meals (group III, n = 11) for a period of 2 months. The drugs were supplied as follows: fenproporex as Desobesi (Asta Medica Laboratories, Sao Paulo, Brazil), sibutramine as Reductil (Knoll Pharmaceutical Co., Ludwigshafen, Germany), and orlistat as Xenical (Hoffman-LaRoche Inc., Basel, Switzerland). Concomitantly, all participants were encouraged to eat a balanced, low-calorie diet of 1200 kcal/d during the whole period of the study.
Measurements
All measurements were obtained at study entry and 2 months after therapy. Body weight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, and BMI was calculated as weight/height2 (kg/m2). Waist circumference was measured in the standing position with a flexible plastic tape at the narrowest part of the torso, as seen from the anterior view (26). The hip girth was measured at the widest part of the hip, and the waist-to-hip circumference ratio was calculated. Total body fat was determined by dual-energy x-ray absorptiometry (DXA) measurements (Lunar Corp. DPX, Madison, WI). In one severely obese woman, DXA was not performed due to technical limitations.
Blood and CSF collection
Blood and CSF collection were carried out at study entry and 2 months after treatment. Venous blood samples were collected after an overnight fast, separated by centrifugation, and stored at -20 C until analysis. Serum leptin concentrations were determined in duplicates in the same run by a commercial RIA (Linco Research, Inc., St. Charles, MO), with a detection limit of 0.5 ng/ml. Coefficients of variation at leptin concentrations between 4.9 and 25.6 ng/ml ranged from 3.4–8.3% within runs and from 3.0–6.2% between runs. Simultaneously to the blood sampling, approximately 4 ml of CSF was obtained by lumbar puncture with patients in lateral decubitus position. The procedure was performed under supervision of a specialized physician (S.M.A.), and no major complications were observed. CSF samples were immediately stored at -20 C until analysis. All CSF samples were assayed in the same run. CSF leptin concentrations were measured in duplicates by a new ultrasensitive commercial assay (Linco Research, Inc.). In the latter assay, the detection limit was 0.05 ng/ml, and the coefficients of variation at leptin concentrations between 0.44 and 4.24 ng/ml ranged from 3.74–7.48% within runs and from 3.24–8.90% between runs.
Statistics
The descriptive statistical results are presented as mean ± SD, unless otherwise stated. One-way ANOVA test and Kruskal-Wallis nonparametric test were used for comparison between groups. Values obtained before and after treatment were compared by using paired t test and Wilcoxon’s matched pairs rank sum test. Correlations were sought by calculating Pearson’s correlation coefficient. A P value less than 0.05 was considered significant.
Results
Table 1
shows the baseline characteristics of the patients. At baseline, the study groups were similar regarding age, weight, BMI, waist circumference, hip girth, total body fat, leptin concentrations in serum and CSF, and CSF/serum leptin ratio. In the whole study group (n = 31), serum leptin levels were positively correlated with BMI (r = 0.58; P < 0.001), waist circumference (r = 0.45; P = 0.012), hip girth (r = 0.54; P = 0.002), total body fat (r = 0.63; P < 0.001) and CSF leptin levels (r = 0.55; P = 0.001). On the other hand, CSF leptin levels were not correlated with any variable. The CSF/serum leptin ratio was negatively associated with BMI (r = -0.37; P = 0.04), body fat percentage (r = -0.38; P = 0.04) and serum leptin (r = -0.46; P = 0.009).
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). In group I, weight loss was 7.6 ± 4.3 kg, and this value was not different compared with the weight reduction in group II (6.3 ± 2.9 kg) and group III (6.6 ± 2.2 kg). Accordingly, BMI, waist circumference, hip girth, and total body fat decreased significantly in each study group, but the changes were similar among the groups (Table 2).
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). When leptin levels were adjusted for the amount of body fat estimated by DXA, there was no difference in the values obtained before and after therapy in each group.
CSF leptin levels were significantly lower after weight loss in all study groups. In group I, the values changed from 0.42 ± 0.11 to 0.31 ± 0.12 ng/ml (P < 0.01), in group II from 0.43 ± 0.15 to 0.24 ± 0.12 ng/ml (P < 0.0001), and in group III from 0.45 ± 0.18 to 0.22 ± 0.08 ng/ml (P < 0.0001). The decline was significantly higher on group III (50.7 ± 12.9%) compared with that in group I (28.2 ± 19.3%; P = 0.006) (Table 2). Individual values of CSF and serum leptin concentrations and CSF/serum leptin ratio at baseline and 2 months after therapy are shown in Fig. 1.
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). In fenproporex-treated women of group I, the ratio was 1.57 ± 0.3% at baseline and 1.72 ± 0.62% after 2 months (P > 0.05), whereas in the sibutramine-treated women of group II, the corresponding values were 1.78 ± 1.01% and 1.69 ± 1.27%, respectively (P > 0.05) (Table 2). On the other hand, in the orlistat-treated women of group III, CSF/serum leptin ratio fell significantly from 1.65 ± 0.43% at baseline to 1.09 ± 0.47% after 2 months (P < 0.01). As shown in Fig. 2, this variation of CSF/serum leptin ratio in group III corresponded to a decrease of 33.3 ± 22.5% in the CSF/serum leptin ratio, which was significantly different from the positive variation in group I (11.9 ± 42.1%; P = 0.006), and close to the statistical significance compared with the negative variation in group II (-7.6 ± 27.8%; P = 0.06).
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The mechanisms of leptin transport into CNS are complex and not completely understood. Leptin binds at both choroid plexus (blood-CSF barrier) and brain endothelial cells (blood-brain barrier), where a high expression of the short form of leptin receptor is found (3, 27, 28). Despite the uncertainty of which is the most important site of brain leptin uptake, there is agreement that leptin efflux occurs via CSF reabsorption, with no evidence for a saturable transport system to remove it from the brain (6, 28). Consequently, changes in CSF leptin levels reflect changes in brain leptin uptake, whether the main leptin uptake occurs at the blood-brain barrier or the blood-CSF barrier. Alternatively, leptin might enter the brain through areas where endothelial barrier is diminished or absent, known as circumventricular organs. One of these areas is represented by the median eminence of the hypothalamus, where a high leptin uptake has been demonstrated (28). However, this mechanism of transport is probably limited due to both a poor diffusion rate of leptin within brain tissue and a mechanical blockade by a layer of epithelial cells of the circumventricular organs (7). Once leptin is stable in blood and CSF, it has poor penetration into brain tissue, and it does not have a saturable transport system out of the brain (6, 28); the estimation of CSF/serum leptin ratio seems a suitable approach to clinically evaluate leptin transport into the brain (5, 6, 7, 10, 11, 22, 23, 24, 25).
In the present study, we have investigated serum, CSF, and CSF/serum leptin ratio in a group of 31 obese women with a mean BMI of 38.2 kg/m2, before and 2 months after a weight loss program consisting of hypocaloric diet and drug therapy for obesity. All participants in our study lost weight, on average 7% of their initial body weight. Serum leptin levels decreased significantly during drug-induced weight loss, with no difference observed among the study groups. The decline in serum leptin was proportional to the changes in body fat, as indicated by leptin levels adjusted to the amount of fat, which were not different before or after weight loss in the groups. Niskanen et al. (29) and Rissanen et al. (30) did not find changes in fat-adjusted levels of leptin after a weight reduction of 9–11% in patients subjected to diet programs for at least 17 wk, alone or in association with orlistat. On the other hand, other studies in which serum leptin was measured in a period of time shorter than 6 wk or in individuals subjected to a caloric restriction of 1000 kcal/d or less, the decline observed in serum leptin levels was higher than that expected only by changes in weight or body fat (31, 32, 33, 34). Therefore, changes in serum leptin levels during weight loss seem to be dependent on severity and duration of caloric restriction, gender, and body fat mass, but are not influenced by antiobesity drugs, as shown by our results.
CSF leptin levels in our obese women were comparable to the values reported by Caro et al. (10) in their group of obese subjects. Leptin concentrations in CSF decreased significantly in all study groups after weight loss, but the decline was more pronounced in the orlistat group than the fenproporex group. In the orlistat-treated women, CSF/serum leptin ratio significantly declined after weight loss, with a mean posttherapy value 33% lower than that from baseline. Because orlistat acts on the intestinal lumen and is minimally absorbed (21), it is unlikely that this decline in CSF/serum leptin ratio was caused by a direct effect of the drug. It is known that high-fat diets lead to a peripheral leptin resistance in rodents (35, 36, 37). Dietary records of our patients did not show differences in the proportion of the intake of each macronutrient among the study groups (data not shown). One should expect a reduction in leptin resistance and higher CSF/serum leptin ratios in the orlistat-treated group if women taking orlistat consumed less fat than those on the other two groups. However, the opposite was found in our study. Therefore, the decrease of CSF/serum leptin ratio during orlistat treatment was likely due to the weight loss per se rather than a direct or indirect effect of the drug.
In mice, Kastin and Akerstrom (38) found that fasting results in a lowered transport of leptin across the blood-brain barrier, as shown by a decrease in CSF/serum leptin ratio, whereas refeeding results in elevated transport of leptin. In humans, there are few clinical studies addressing serum, CSF, and CSF/serum leptin ratio during therapy for obesity. In a study of obese women evaluated before and after 2 wk of a very low-calorie diet (586 kcal/d), Krotkiewski et al. (25) showed that CSF leptin levels fell around 50%, a value similar to that seen in our orlistat-treated women, but higher than the 28% decrease in CSF leptin levels observed during therapy with fenproporex. In the same study, the authors showed an increment in CSF/serum leptin ratio after 2 wk of weight loss induced by a very low-calorie diet. In contrast, we found a significant decrease in CSF/serum leptin ratio in women treated with orlistat. Taken together, these findings suggest that the leptin transport system might be distinctively affected by acute and chronic caloric restriction and the degree of weight loss in obese women.
In our study, CSF/serum leptin ratio did not change after weight loss in women treated with centrally acting drugs. A possible explanation for the maintenance of the CSF/serum leptin ratio during therapy with fenproporex and sibutramine is that adrenaline and noradrenaline enhance leptin transport across the blood-brain barrier by about 2- to 3-fold, working through the 
-adrenergic receptors (39). This effect seems to be specific, because other monoamines such as serotonin and acetylcholine did not affect the entry of leptin into the brain (7, 39). Therefore, one of the mechanisms by which noradrenergic drugs such as fenproporex and sibutramine inhibit food intake might be due to the maintenance or increment of leptin transport into the brain.
In conclusion, our results showed a decrease in CSF/serum leptin ratio after weight loss in obese women treated during 2 months with orlistat, whereas this ratio did not change significantly during therapy with fenproporex and sibutramine. These findings potentially reflect an interference of these centrally acting antiobesity drugs in leptin transport across the blood-brain barrier, although other mechanisms influencing CSF/serum leptin ratio might also explain these changes.
Acknowledgments
We are grateful to Asta Medica Laboratories, Knoll Pharmaceutical Co., and Hoffman-LaRoche Inc. for kindly providing the drugs used in the study.
Footnotes
This work was partly supported by grants from the Fundo de Desenvolvimento Academico of the Universidade Federal do Parana, Brazil.
Abbreviations: BMI, Body mass index; CNS, central nervous system; CSF, cerebrospinal fluid; DXA, dual-energy x-ray absorptiometry.
Received August 14, 2001.
Accepted January 11, 2002.
References
1-adrenergic agents. Brain Res 899:209–217[CrossRef][Medline]This article has been cited by other articles:
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  K. Kos, A. L. Harte, N. F. da Silva, A. Tonchev, G. Chaldakov, S. James, D. R. Snead, B. Hoggart, J. P. O'Hare, P. G. McTernan, et al. Adiponectin and Resistin in Human Cerebrospinal Fluid and Expression of Adiponectin Receptors in the Human Hypothalamus J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1129 - 1136. [Abstract] [Full Text] [PDF]
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, Group I, fenproporex (n = 10); 
, group II, sibutramine (n = 10); 
, group III, orlistat (n = 11). Horizontal lines show mean values for the whole group (n = 31). Asterisks indicate P < 0.0001 vs. baseline.