Weight Loss Increases Soluble Leptin Receptor ... - Wiley Online Library

Weight Loss Increases Soluble Leptin Receptor Levels and the Soluble Receptor Bound Fraction of Leptin Markus Laimer,* Christoph F. Ebenbichler,* Susanne Kaser,* Anton Sandhofer,* Helmut Weiss,† Hermann Nehoda,† Franz Aigner,† and Josef R. Patsch*

Abstract LAIMER, MARKUS, CHRISTOPH F. EBENBICHLER, SUSANNE KASER, ANTON SANDHOFER, HELMUT WEISS, HERMANN NEHODA, FRANZ AIGNER, AND JOSEF R. PATSCH. Weight loss increases soluble leptin receptor levels and the soluble receptor bound fraction of leptin. Obes Res. 2002;10:597– 601. Objective: Soluble leptin receptor (sOB-R) represents the main binding site for leptin in human blood. The aim of this study was to investigate the relationship between leptin and soluble leptin receptor and the bound/free ratio after pronounced weight reduction. Research Methods and Procedures: A total of 18 morbidly obese women participated in this prospective study. Subjects were examined for fat mass, leptin, and sOB-R concentrations before and 1 year after Swedish adjustable gastric banding. Results: Anthropomorphic measures displayed a significant reduction of body mass index [(42.9 ⫾ 5.6 to 32.9 ⫾ 6.0 kg/m2 (mean ⫾ SD)]. Fat mass decreased from 56.3 ⫾ 9.0 to 33.9 ⫾ 12.5 kg. Plasma leptin concentration decreased from 44.6 ⫾ 18.0 to 20.0 ⫾ 13.1 ng/mL (p ⬍ 0.001), whereas the sOB-R levels increased from 11.1 ⫾ 3.6 to 16.6 ⫾ 6.0 U/mL after weight-reducing surgery. Thus, the sOB-R bound fraction of leptin increased from 7% to 33%. Discussion: This work demonstrates a relationship between weight loss, leptin, and sOB-R concentrations in vivo. During weight loss, leptin levels decreased, whereas sOB-R

Submitted for publication November 13, 2001. Accepted for publication in final form March 8, 2002. *Department of Medicine and †Department of General Surgery, University of Innsbruck, Austria. Address correspondence to Christoph F. Ebenbichler, M.D., Universita¨tsklinik fu¨r Innere Medizin, Universita¨t Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. E-mail: [email protected] Copyright © 2002 NAASO

levels and the receptor bound fraction of leptin increased. Thus, sOB-R may negatively regulate free leptin. Key words: soluble leptin receptor, leptin, weight-reducing surgery, Swedish adjustable gastric banding

Introduction The hormone leptin and its receptor (OB-R), a member of the class I cytokine receptor family, are key homeostatic regulators of mammalian body weight. Leptin is predominantly secreted by adipocytes (1). Circulating leptin is found in human serum either as a free form of a 16-kDa protein or as a bound form (2). Leptin carrier proteins have been described to correspond with the size of the extracellular domain of the leptin receptor (2– 4). Similar to OB-R, other class I cytokine receptors produce soluble receptors corresponding to the extracellular domain of the receptor (5). The soluble OB-R (sOB-R) is the major binding protein in the circulating human blood (6). In ob/ob mice, which have no endogenous leptin, sOB-R overexpression leads to an increase in the weight-reducing effect of administered leptin (7). In humans, obesity is associated with elevated plasma leptin concentrations (8,9). Interestingly, the majority of leptin circulates as free leptin, whereas in lean subjects, the majority of leptin circulates in bound form (2). The aim of this study was to determine prospectively whether pronounced weight reduction influences leptin and sOB-R concentrations and the bound/free ratio of leptin.

Research Methods and Procedures Subjects Eighteen morbidly obese women (body mass index ⬎ 40 kg/m2) participated in this prospective study. Exclusion criteria were secondary causes of adiposity, diabetes mellitus, pregnancy, intake of lipid-lowering drugs, or another OBESITY RESEARCH Vol. 10 No. 7 July 2002

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Soluble Leptin Receptor after Weight Loss, Laimer et al.

Table 1. Baseline characteristics

Age (years) Height (cm) Weight (kg) BMI (kg/m2) Fat mass (kg) Leptin (ng/mL) sOB-R (U/mL)

Pre-SAGB (mean ⴞ SD)

Post-SAGB (mean ⴞ SD)

p Value*

Control subjects (mean ⴞ SD)

p Value†

p Value‡

40.3 ⫾ 9.8 165 ⫾ 6 117.1 ⫾ 15.3 42.9 ⫾ 5.6 56.3 ⫾ 9.0 44.6 ⫾ 18.0 11.1 ⫾ 3.6

41.4 ⫾ 10.2 165 ⫾ 6 89.7 ⫾ 15.6 32.9 ⫾ 6.0 33.9 ⫾ 12.5 20.0 ⫾ 13.1 16.6 ⫾ 6.0

⬍0.001 n.d. ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

38.3 ⫾ 9.8 166 ⫾ 6 63.1 ⫾ 6.9 22.9 ⫾ 2.2 15.9 ⫾ 3.2 6.3 ⫾ 3.3 19.5 ⫾ 2.7

n.s. n.s. ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

n.s. n.s. ⬍0.001 ⬍0.001 ⬍0.001 0.01 n.s.

* p values are given for the comparison of the pre- and post-SAGB subjects by paired-samples Student’s t test. † p values are given for the comparison of the pre-SAGB subjects and the control group by independent-samples Student’s t test. ‡ p values are given for the comparison of the post-SAGB subjects and the control group by independent-samples Student’s t test. SAGB, Swedish adjustable gastric banding; n.s., not significant; n.d., not determinable; BMI, body mass index; sOB-R, soluble leptin receptor.

medically significant illness. Examinations of the study subjects were done within 2 months before Swedish adjustable gastric banding (SAGB) and 1-year post-SAGB. In addition, eight lean female subjects were examined as a control group. Informed consent was obtained before entering into the study, and all procedures were performed in accordance with institutional guidelines at the Internal and Surgical Department of the Medical Faculty of the University of Innsbruck. Surgical Procedure The surgical procedure was performed as described by Forsell et al. (10) at the General Surgical Department of the University Hospital in Innsbruck (11). The SAGB was used in all of the study patients (SAGB Obtech Medical AG, Zug, Switzerland). Analysis of Body Composition Body composition was determined by impedance analysis using a multifrequency body impedance 2000-M analyzer (Data Input, Hofheim, Germany). Fat-free mass and fat mass were determined using Nutri 4 software (Data Input). Laboratory Measurements Blood was drawn after an overnight fast from an antecubital vein into EDTA tubes (1.6 mg/mL). Immediately after collection, plasma was separated from erythrocytes by centrifugation at 3000 rpm for 10 minutes at 4 °C. Plasma samples were stored frozen at ⫺80 °C until assayed. Leptin was measured using an enzyme-linked immunosorbent assay kit (R&D Systems, Wiesbaden, Germany). 598

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sOB-R concentration was measured using a sOB-R enzyme-linked immunosorbent assay kit (Chemicon International, Temecula, CA). The sensitivity of the assay is 0.4 U OB-R/mL and the range of detection is 2 to 100 U OB-R/ mL. According to the manufacturer of the assay, 1 U/mL OB-R approximates 2 ng of native human sOB-R per milliliter. Statistical Analysis Descriptive data are expressed as mean value ⫾ SD. The data from pre- and post-SAGB subjects were compared using a paired-samples Student’s t test. Linear association between the ⌬ values of variables under investigation were calculated using the method of Pearson’s correlation coefficient. Statistical significance was inferred at a two-tailed p value of ⬍ 0.05. Statistical analyses were calculated using SPSS release 8.0 for Windows (SPSS, Chicago, IL).

Results Demographic measures are displayed in Table 1. Leptin levels decreased from 44.6 ⫾ 18.0 ng/mL in pre-SAGB subjects to 20.0 ⫾ 13.1 ng/mL in post-SAGB subjects (p ⬍ 0.001, Figure 1A). The sOB-R concentrations increased from 11.1 ⫾ 3.6 U/mL (22.2 ⫾ 7.2 ng/mL) before SAGB to 16.6 ⫾ 6.0 U/mL (33.2 ⫾ 12.0 ng/mL) after the banding procedure (p ⬍ 0.001, Figure 1B). The calculated bound fraction of leptin increased from 7 ⫾ 6% before SAGB to 33 ⫾ 31% after SAGB (p ⫽ 0.002, Figure 1C). Correlation analysis of the ⌬ values displayed a positive relationship between leptin decrease and weight loss (r ⫽ 0.579, p ⫽ 0.012). In contrast, the sOB-R increase correlated negatively with weight reduction (r ⫽ ⫺0.641, p ⫽


Figure 1: (A) The mean leptin concentration (⫾SEM) of 18 female subjects before and 1 year after gastric banding (p ⬍ 0.001). (B) In contrast, soluble leptin receptor (sOB-R) increased significantly after pronounced weight loss (p ⬍ 0.001). (C) The calculation of the bound fraction of leptin revealed a significant increase of bound leptin from 7% to 33% after gastric banding (p ⫽ 0.002).

0.004). Furthermore, an inverse relationship was found between leptin decrease and sOB-R increase in a partial correlation analysis, adjusted for the influence of age and obesity (r ⫽ ⫺0.512, p ⫽ 0.04).

Discussion The adipocyte-derived hormone leptin exerts its effects through the OB-R, a cytokine class I receptor (12).

In mice, several isotypes of this receptor are alternatively spliced. OB-Ra is the predominant form expressed in most tissues, including choroid plexus and brain microvessels (13,14). Although OB-Ra is expressed most abundantly, this OB-R isoform is not capable of signal transduction. In contrast, OB-Rb is highly expressed in the hypothalamus (15), and this OB-R isoform exerts full-signaling capacity (12). OB-Rb is responsible for mediating the central effects of leptin on appetite and energy expenditure (16). A third alternatively spliced OB-R isoform has been identified; it lacks the intracellular and transmembrane domains of OB-Rb (17). OB-Re is thought to be the circulating, leptinbinding isoform of the OB-R family (7). In humans, two isoforms of OB-R have been described. OB-RL (similar to OB-Rb in the mouse) is the isoform with full-signaling capacity, whereas OB-Rs (similar to OB-Ra in mouse) lacks the domains capable of signal transduction (18). A characteristic of this cytokine-receptor family is the production of soluble receptors, which circulate and bind their respective ligand (5). Soluble receptors can be produced by both proteolytical cleavage and alternative splicing (5,19,20). The soluble growth hormone receptor is produced in a species-specific manner (21–23). In addition, the growth hormone– binding protein is a dominant negative regulator of the growth hormone receptor (24,25). Thus, the soluble receptor isoforms may be important regulators of the ligands’ action on the respective, signaling-competent receptor isoforms. Regulation of the sOB-R concentrations has been described for several conditions. sOB-R concentrations are modulated throughout the life cycle: levels are low at birth, high in prepubertal age, and then fall and remain stable throughout adult life (26). sOB-R increases during pregnancy and presents differently in diabetic and nondiabetic women (27). In obese patients, low levels of circulating OB-R were reported when compared with lean control subjects (2). Short-term fasting induces a decline in free and bound leptin in lean and obese women, with more pronounced relative changes in lean subjects. The equilibrium between free and bound leptin fractions was not influenced during short-term fasting (28). The hypothalamus is thought to be the major target for leptin. In cerebrospinal fluid, only leptin in its free form, devoid of sOB-R, is detected, suggesting that the free form of leptin is the biologically active form (29). In this study, we prospectively examined women undergoing weight loss induced by surgical intervention. As expected, we observed a marked reduction of leptin. In contrast, the sOB-R concentrations increased. When calculating the bound fraction of leptin to the sOB-R, we observed a significant increase of bound leptin after weight reduction. Correlation analysis revealed, as expected, a strong positive correlation between weight loss and leptin OBESITY RESEARCH Vol. 10 No. 7 July 2002

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decrease. In contrast, sOB-R concentrations correlated negatively with weight reduction. Weight reduction induced by bariatric surgery is associated with complex hormonal changes (30). These hormonal changes, such as variations in insulin levels, may play a role in the regulation of sOB-R levels. Recent evidence suggests that sOB-R is produced by proteolytic cleavage of the membrane-spanning isoforms in humans. Thus, the occurrence of the sOB-R may be regulated in humans by membrane-bound protease (31). The density of membrane-bound proteases could be responsible for this inverse relationship between leptin and sOB-R. This prospective study reveals a relationship between weight reduction, leptin, and sOB-R concentrations in vivo. During weight reduction, leptin levels decreased, whereas sOB-R levels and the receptor bound fraction of leptin increased. Thus, sOB-R may negatively regulate free leptin.

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