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Secretion of Free and Protein-Bound Leptin from Subcutaneous Adipose Tissue of Lean and Obese Women

Abteilungen Klinische Endokrinologie (G.B., B.M.), Anatomie (H.N.), Viszeral and Transplantationschirurgie (M.B.), Medizinische Hochschule Hannover, 30623 Hannover, Germany; and Department of Medicine, Karolinska Institute Huddinge University Hospital (V.v.H., P.A.), S14186 Huddinge, Sweden

Address all correspondence and requests for reprints to: G. Brabant, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, Carl Neubergstrasse 1, D-30623 Hannover, Germany. E-mail: . brabant.georg{at}mh-hannover.de

Abstract

Leptin circulates as a free (FL) and a protein-bound (BL) form, with the soluble leptin receptor (LR) as an important binding compound. Here we measured these components of leptin in serum and in the incubation medium of sc adipose tissue in healthy lean (n = 10) and obese (n = 13) female subjects using recently developed specific RIA systems. In addition, immunostaining for FL, BL, and LR in adipose tissue was performed. Serum FL levels were increased in the obese subjects (P < 0.0001), whereas BL and LR concentrations in serum of lean and obese subjects were similar. Both FL and BL were secreted from human preadipocytes and increased in parallel to the differentiation of the cells. In sc fat cell explants LR antibodies predominantly stained the fat cell membrane, whereas FL and BL antibodies revealed intracytoplasmatic adipocyte staining. The release of FL, BL, and LR from adipose tissue was increased in obese compared with lean subjects (P < 0.005 for FL; P < 0.02 for BL, and P < 0.01 for LR). In summary, fat cells are capable of releasing not only FL, but also BL and LR.

LEPTIN, AN ADIPOCYTE-DERIVED hormone, is believed to be a major regulator of food intake and energy homeostasis in man (1, 2). Direct evidence for the physiological importance of leptin in humans has recently been provided in a leptin-deficient child who dramatically lost weight after leptin replacement therapy with low doses of the hormone (3). These effects are mediated via central hypothalamic feedback mechanisms, but, in addition, a large range of peripheral leptin actions in different subsystems have been discussed, including feedback effects on pancreatic ß-cell function and liver and muscle cells (4, 5, 6, 7). Physiological leptin effects are mediated via interaction of leptin with specific leptin receptors (LR) expressed in many tissues (8, 9). Several isoforms of the receptor have been characterized (8, 9). The shortest isoform lacking the hydrophobic transmembrane domain codes for a soluble, secreted variant capable to bind free leptin (FL) (10, 11, 12). In serum, leptin circulates as free hormone and in high molecular weight complexes formed with its soluble receptor even though binding to other proteins may exist as well (13, 14, 15). Recent evidence reported by our group suggests that bound leptin (BL) serves as a marker of resting energy expenditure, whereas FL selectively reflect body fat mass (16, 17, 18). As FL and BL are differentially regulated in various pathophysiological conditions, BL may serve independent functions (17, 18, 19). The concept of a different function of a receptor-bound cytokine has recently been postulated for other closely related cytokines such as IL-6 and may serve as a general principle in the activation of cytokines signaling via dimer receptors (19, 20).

Currently it is unknown how and where BL is formed. Studies in fat cells indicate that adipocytes not only synthesize and secrete leptin, but also express leptin receptor isoforms (1, 2, 14, 21, 22, 23, 24). In these studies total leptin and the leptin receptor were determined, but it remains unclear whether FL and BL are present in adipose tissue. Thus, BL may be formed and secreted by the adipose tissue, or it may be formed from FL and LR in the circulation or in tissue(s) outside adipose tissue. In the present study we attempted to better delineate the origin of leptin components by comparing the release of LR, FL, and BL from adipose tissue to the circulating serum levels in lean and obese subjects using recently developed specific RIA systems (25, 26, 27).

Subjects and Methods

Subjects

Ten lean and 13 obese female subjects volunteered for the study. After extensive explanation of the study protocol, informed consent was obtained from all subjects. The study protocol was approved by the local committee of medical ethics. All subjects were healthy, free of any medication, and premenopausal. They were investigated approximately 2 wk before expected start of the menstrual period. None was completely sedentary or involved in athletic performances. None had undertaken a recent weight loss program. Obesity was defined as a body mass index (BMI) greater than 30 kg/m². The clinical characteristics of both groups are given in Table 1.

In the second part of the investigation sc fat was obtained from six subjects (three men and three women; 56–70 yr of age; BMI, 22–38.3 kg/m²) and used for methodological studies, immunohistochemistry, or preadipocyte experiments. Those subjects underwent elective surgery for nonmalignant disorders (gallstones, hysterectomy, or gastric banding for obesity). Tissue was removed from the surgical incision at the beginning of operation.

Experiments with adipose tissue fragments

Experiments were performed as exactly described previously (29). Briefly, one part of the tissue was used for the preparation of isolated adipocytes and subsequent determination of fat cell size and number. The second part (300 mg) was incubated with air as the gas phase for 2 h at 37 C in a Krebs-Ringer phosphate buffer containing 20 mg/liter BSA and 1 g/liter glucose. An aliquot (3 ml) of the medium was stored at -70 C for subsequent analysis of LR, FL, and BL. The incubated adipose tissue was subjected to organic extraction, and total lipid content was determined gravimetrically. The release of LR, FL, and BL into the medium was related both to the lipid content of incubated adipose tissue and to the total number of fat cells. Methodological experiments revealed that the release of FL and BL was linear over an incubation time of at least 4 h.

Culture of human preadipocytes

Subcutaneous adipose tissue (10 g) was obtained from three obese women during gastric banding for obesity. Tissue was taken from the surgical incision at the beginning of surgery and was immediately brought to the laboratory in saline. The isolation and differentiation of preadipocytes were performed according to the method described by Hauner et al. (30). Fragments of approximately 5–10 mg were used and incubated for 1 h at 37 C in a shaking bath with 0.5 g/liter collagenase (Sigma, St. Louis, MO) in Krebs-Ringer phosphate buffer (pH 7.4) with 40 g/liter dialyzed BSA (fraction V, Sigma). A cell suspension was incubated in 10 ml erythrocyte lysis buffer consisting of 0.154 M NH₄Cl, 5.7 mM K₂HPO₄, and 0.1 mM EDTA (pH 7.3) for 10 min at room temperature. The preadipocyte fraction was inoculated into 24-well plates at a density of 50,000 cells/cm² and kept at 37 C in 5% CO₂ for 20 h. Then the cells were washed twice with DMEM/nutrient mixture F-12 medium with 100 nM cortisol, 66 nM insulin, 15 mM HEPES, 1 nM T₃, 33 µM biotin, 17 µM panthothenate, 10 µg/ml transferrin, 100 µg/ml penicillin-streptomycin, 2.5 µg/ml amphotericin B, and 10 µM of the thiazolidinedione BRL 49653, which is a peroxisomal proliferator-activated receptor- agonist (provided by SmithKline Beecham Pharmaceuticals, Harlow, UK). After 2–3 d of incubation, cell differentiation was first evaluated by inspection through a microscope and counting of differentiated adipocytes. Incubation plates containing more than 5% endothelial cells were discarded. Then, incubation medium was removed and kept at -70 C for subsequent analysis of leptin components. Thereafter differentiation was reexamined by determining the lipid content of cells after Oil Red O staining.

Assays of leptin parameters

Serum, incubation medium of the adipose explants, and supernatant culture medium from preadipocytes were transported frozen over dry ice to Hannover for measurements of FL, BL, and LR. All assays have been previously described in detail (16, 25, 26, 27). In short, we generated antibodies to the N-terminal portion of the protein (leptin amino acids 26–38) and to a C-terminal fragment (leptin amino acids 126–140) by coupling to hemocyanin by the carbodiimide method. N-Terminal antibodies selectively detect BL immunoreactivity, whereas C-terminal antibodies selectively reflect FL. In addition, a midregion part of the extracellular portion of the LR (LR amino acids 364–385) was used for measuring soluble LR by specific antibodies. For assays of the supernatant of fat cell explants, standards were prepared in medium. The maximal cross-reactivity between the different leptin assays (BL and FL, and LR) was less than 0.001.

Immunohistochemistry

Subcutaneous fat specimens were immediately frozen and stored at -80 C. Subsequently, the sc fat specimens were fixed in 4% formaldehyde for 24 h at 4 C and embedded in paraffin. Routine immunohistology was performed. In brief, deparaffinized, lightly dried sections (8 µm thick) were incubated with the primary antibodies against human FL, BL, and LR at a dilution of 1:100 in 5% goat serum. Binding of the primary antibody was revealed by mouse IgG for 30 min. To visualize the binding, an alkaline phosphatase-antialkaline phosphatase complex (DAKO Corp., Carpenteria, CA; 1:50 in TBS for 30 min) was used, followed by repetition of the last two steps for 15 min. The sections were stained with Fast Red for 25 min, counterstained with hematoxylin (1:5 in PBS) for 90 sec, and mounted in Glycergel (DAKO Corp.). Control staining was carried out by omitting the primary antibody.

Statistical evaluation

The values for FL, BL, and LR were not normally distributed. Therefore, we logarithmically transformed both parameters before statistical analysis. Significance was tested by t test and Wilcoxon’s signed rank test. Both tests gave essentially the same information. Linear regression analysis and ANOVA were also performed. P < 0.05 was regarded as significant. All calculations were performed using the SAS Institute, Inc. (Cary, NC) statistical software package.

Results

Clinical characteristics

The clinical characteristics of the volunteers are summarized in Table 1. As expected, BMI and percent body fat were increased in the obese women. This group also showed features of insulin resistance and dyslipidemia, which are typical for obese subjects.

Circulating serum leptin and LR levels

Serum concentrations of FL were markedly and significantly higher in obese subjects compared with lean controls (8-fold higher in obese than in lean), whereas serum BL levels were not significantly influenced by obesity (Table 2). Serum FL concentrations in both lean and obese subjects were closely related to body fat (overall serum FL levels to percent body fat; r² = 0.73; P < 0.0001) contrasting with a nonsignificant relation of serum BL levels (r² = 0.01; P = NS). In both groups LR levels were comparable (Table 2). In lean subjects there was approximately 2.8 times more serum BL than FL (P = 0.002). In contrary, in obese subjects serum FL and BL concentrations were not significantly different (P = NS).

After short incubation of adipose tissue, FL, BL, and LR were detected in the incubation medium, suggesting that all components of the leptin system are released by the adipose tissue. This was further substantiated by the lack of any time-dependent effect on the proportional release of FL and BL into the medium during the incubation period (see Subjects and Methods). The release of LR, FL, and BL was significantly higher in obese than in lean subjects (Table 2). In both lean and obese subjects, adipose tissue secreted significantly more FL than BL (P < 0.004; paired analysis was possible in 15 pairs).

Preadipocyctes cultures

The exposure of sc preadipocytes to a defined differentiation medium resulted in a 6- to 10-fold increase in the release of FL and BL in the supernatant on d 7 of differentiation compared with that on d 3.5. Three different experiments were performed, giving similar results The result of one typical experiment is shown in Fig. 1. Microscopic examination as well as Oil Red O staining revealed a marked increase in fully differentiated adipocytes during this time interval (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 1. Release of FL and BL to supernatant of cultured sc preadipocytes. Cells were differentiated for 3.5 and 7 d, respectively. Three experiments were performed; a typical experiment is depicted. Values are the mean of duplicate incubations.

To elucidate the cellular locations of LR, FL, and BL, we performed immunohistochemistry of sc fat tissue using antibodies directed to FL, BL, and LR. As shown in Fig. 2, staining of fat cells with FL and BL antibodies clearly demonstrated intracytoplasmatic staining, suggesting an intracellular location of immunoreactive material. In contrast, anti-LR antibodies predominantly stained the membranes of the cells. In all cases specific immunostaining was completely blocked by coincubation with the peptides used for generating the respective antibodies (data not shown).

View larger version (60K): [in this window] [in a new window]   Figure 2. Immunostaining of sc adipose tissue for FL (a), BL (b), and LR (c). Magnification, x100. For BL a higher magnification of x400 was used in the inset to better demonstrate intracytoplasmatic BL staining. C, Cytoplasm; M, plasma membrane; N, nucleus.

Leptin is circulating in free and protein-bound forms (12, 13, 15, 16, 17, 31). To date, it has been unclear whether BL is formed in the circulation or is directly synthesized within adipose tissue. Here we show that free and bound leptin are already detectable in supernatants of preadipocyte cultures. Even though experimental conditions used for preadipocytes and those for fat explants differ considerably, and thus direct comparison is difficult, the data are consistent with an increase in leptin and further differentiation of the cells to mature adipocytes. Explants of mature fat cells release FL and LR, which could be detected in the incubation medium. BL is also detectable in the incubation medium of fat explants. Principally bound leptin may be formed by fat cells or outside the adipocytes. From our experiments it is impossible to exclude formation of bound leptin by components of the stroma. However, two lines of evidence favor direct release of BL over formation of BL by the stroma or in the medium. When comparing the increases in FL and BL in methodological experiments over a 4-h time period, a proportional accumulation of both parameters is observed in the medium. Moreover, direct immunostaining of the cells using the respective antibodies directed against FL and BL revealed intracytoplasmatic staining such as obtained with pan-antileptin antibodies (14), indicating a cellular origin of at least part of the released bound form.

Body fat is related to FL secretion rates from adipose tissue. In studies measuring total leptin, increased total leptin secretion from the enlarged fat cells in the obese state was anticipated (1, 2, 21, 24, 29). In our study we show for the first time that there all leptin components, LR, FL, and BL, are released from fat cell explants, and this is increased with obesity.

With regard to circulating leptin concentrations, LR and BL were not influenced by obesity, but FL was markedly increased. In gel chromatographic studies by Shina et al. (13), a clear shift from BL as the dominant circulating form of leptin in the lean to FL in the obese was evident. Here we confirm this idea by specific RIAs, and the results support recent data for nonobese and obese subjects (32).

It is of interest to compare circulating and adipocyte- derived leptin. Subcutaneous adipose tissue secreted FL and BL in the same proportion in lean as in obese subjects, whereas the proportion of FL in relation to BL in the circulation was decreased in the lean state. This suggests that in nonobese subjects BL in addition to a direct release from adipocytes may be actively formed outside the sc fat cells. However, caution should be exercised when comparing leptin release from adipose tissue in vitro with circulating leptin in vivo. In humans leptin circulates bound to several serum macromolecules (12, 31, 33). Whether these binding proteins are the same as those derived from adipose tissue remains to be established. It should also be stressed that adipocytes may not be the only source of BL within adipose tissue. Although our results clearly demonstrated that formation of BL takes place within the adipocytes, it is possible that stromal cells also contribute to the release of BL from adipose tissue. Even though a quantitative contribution of other adipose regions appears less likely, because the secretion rate of total leptin is much lower from visceral than sc adipose tissue, the potential role of other fat depots remains to be established (29, 34). Moreover, an important contribution of other metabolically active organ systems rich in LR, such as liver or muscle, needs further investigation.

In conclusion, FL, BL, and LR are directly released from sc adipose tissue, and their release is increased in obesity. It is proposed that the formation of BL by adipose tissue may represent an important component in the regulation of free vs. bound leptin in the circulation.

Acknowledgments

We are grateful for the advice of H. Hauner when setting up the preadipocyte system, and for R. Horn’s help and technical assistance.

Footnotes

This work was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Foundation, the Novo Nordisk Foundation, and the Swedish Heart and Lung Association.

Abbreviations: BL, Protein-bound leptin; BMI, body mass index; FL, free leptin; LR, leptin receptor.

Received September 19, 2001.

Accepted May 3, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints, Permissions and Rights Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brabant, G. Articles by Arner, P. Search for Related Content PubMed PubMed Citation Articles by Brabant, G. Articles by Arner, P.