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Original Studies |
Department of Medicine, Santiago de Compostela University, Endocrine Section (X.C., V.P., R.P., M.L., J.C., F.F.C.), the Department of Morphological Sciences (R.G.), and the Department of Physiology (C.D.), University of Santiago de Compostela; and the Division of General Surgery (L.G.V.), Hospital de Conxo, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: F. F. Casanueva, M.D., Ph.D., P.O. Box 563, Santiago de Compostela E-15780, Spain. E-mail: meffcasa{at}uscmail.usc.es
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Abstract |
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To clarify these points, a systematic study with organ culture from human omental adipose tissue either stimulated or not with steroid hormones was undertaken in samples obtained at surgery from 67 nonobese donors (33 women and 34 men). The assay was standardized in periods of 24 h ending at 96 h, with no apparent tissue damage. Each adipose tissue sample from a single donor was incubated in triplicate, and leptin results are expressed as the mean ± SEM of the integrated secretion to the medium (area under the curve; nanograms of leptin per g tissue/48 h).
Control nonstimulated samples showed a steady leptin secretion along the 96 h studied, with the peak of secretory activity reached at 48 h; afterward, the in vitro secretion reached a plateau state. Spontaneous leptin secretion in samples from 33 women (3904 ± 347) was significantly higher (P < 0.05) than that in samples from 34 men (2940 ± 323). Coincubation of adipose tissue with 1 µmol/L dexamethasone induced a clear-cut leptin increase (P < 0.05) in samples from women (5848 ± 624; n = 12), but did not change the spontaneous release of leptin in samples from men (3353 ± 741; n = 6). Similarly, coincubation of adipose tissue with 1 µmol/L estradiol induced a notable leptin increase (P < 0.05) in samples from women (5698 ± 688; n = 9), whereas it did not alter the secretion in the male samples (3373 ± 444; n = 6). In samples from both sexes, coincubation with 1 µmol/L estrone or progesterone had no effect, whereas 1 µmol/L forskolin significantly (P < 0.05) reduced leptin release.
In conclusion, leptin secretion from omental adipose tissue in vitro 1) is significantly higher in samples from women than in samples from men, 2) is stimulated by dexamethasone and estradiol in women but not in men, 3) is not modified by progesterone or estrone in both sexes, and 4) is inhibited by forskolin in both genders. This different response to the stimulation of adipose tissue may be the biological basis for the gender differences observed in circulating levels of human leptin.
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Introduction |
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There are remarkable gender differences in circulating leptin levels, with leptin concentrations in women being nearly twice as high as those in men (35, 36, 37, 38). These gender differences are observed even when subjects from opposite sex were compared after being carefully matched for the net amount of fat reserves (13, 39) and for stages of development, such as peripuberty (19, 40) or birth (41, 42). As no sex-based differences in the adipose tissue reserve are expected in those studies, the existence of and the physiological basis for a gender difference in serum leptin concentrations in man are still open questions.
In the present work, leptin secretion by human omental adipose tissue obtained at surgery was studied in vitro with three aims: 1) to understand the biological basis of the gender differences in human leptin secretion; 2) to assess the real role of relevant steroid hormones, such as glucocorticoids, estrogens, and progesterone, in leptin release; and 3) to validate the in vitro system as a useful tool for further studies.
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Materials and Methods |
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Excised adipose tissue was immediately transported to the laboratory in ice-cold Krebs-Ringer-HEPES buffer (NaCl, 125 mmol/L; KCl, 5 mmol/L; MgSO4, 1.2 mmol/L; CaCl2, 2 mmol/L; KH2PO4, 2 mmol/L; glucose, 6 mmol/L; HEPES, 25 mmol/L; pH 7.4). After removing blood vessels and conjunctive tissue, adipose tissue was washed with sterile Krebs-Ringer-HEPES and cut into small pieces with sharp scissors. Tissue fragments were placed in six-well dishes (300–400 mg adipose tissue/well) containing 2.5 mL DMEM plus 0.5% FCS, supplemented with penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL). In the experiments with estradiol, phenol red-free DMEM was used to avoid interferences of the dye. After a preincubation period of 1 h at 37 C under a humidified atmosphere of 95% air-5% CO2, the media were aspirated, and 2.5 mL fresh medium (with or without stimuli) were dispensed into each well. Culture media were then collected every 24 h and replaced with fresh medium, again with or without stimuli. The drugs tested were dexamethasone, forskolin, estradiol, estrone, and progesterone, all at 1 µmol/L, with the appropriate vehicle added to the control samples. Such concentrations were selected after dose-response studies as the most able to be active in the organ culture system used. Unless specifically indicated, all drugs and reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
For each tested variable, either untreated or treated sample, the adipose tissue from a given subject was independently incubated in triplicate, and the medium was collected and analyzed every 24 h to obtain the 24-h secretion, the cumulative secretion until 96 h, and the integrated secretion [area under the curve (AUC)]. Samples were stored at -20 C until assayed for leptin secretion. Serum leptin levels were measured in duplicate by RIA using commercial kits (Human Leptin RIA, Linco Research, St. Charles, MO). The limit of sensitivity was 0.5 µg/L, the intraassay coefficient of variation was 8.3%, and the interassay coefficient of variation was 6.2%.
Histological analysis
Samples of abdominal adipose tissue were fixed by immersion in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4, for 3 h, washed in the same buffer, and postfixed in 2% osmium tetroxide in veronal acetate buffer (0.056 mol/L; pH 7.4) for 1 h. Inclusion was performed in Spurr’s epoxy resin (43). Semithin sections (1 µm thick) were stained with 1% toluidin blue. Codified samples were processed in a single blind fashion, and representative fields were photographed for morphometry.
Morphometric analysis was performed after digitalization of the pictures. Images were processed on a Macintosh LC475 computer using the public domain NIH Image analysis program (version 1.59, written by Wayne Rasband at the NIH and available from the internet by anonymous FTP from zippy.nimh.nih.gov, or on floppy disk from the National Technical Information Service, Springfield, VA, part PB93-504868). After opening each image, differences in background lightening were eliminated by means of automatic background subtraction (using the two-dimensional rolling ball algorithm), and cell contours were isolated by manually selecting a density slice containing the cell boundaries but not the cytoplasm. Next, every cell was selected, and simultaneous measurements of cross-sectional area and perimeter were obtained. The system was previously calibrated using a micrometric grid photographed at the same magnification, and for each individual, 150–300 cells were counted.
Statistical analysis
The mean BMI, defined as weight in kilograms divided by the square of height in meters, was calculated. Data are presented as the mean ± SEM of the group. Leptin secretion was expressed as the total amount of leptin secreted to the well by a given sample (in nanograms) divided by the amount of fat tissue of the sample in grams, i.e. nanograms of leptin per g tissue (44). The AUC was calculated by the trapezoidal method and is expressed as nanograms of leptin per g tissue/48 h. Comparisons were made using the unpaired t test. The correlation between two variables was assessed by Pearson’s coefficient of correlation. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Afterward, a slow reduction was
observed up to the longer studied times (144 h; data not shown). For
this reason, all studies were limited to 96 h. Neither the
histological analysis nor the biochemical analysis of leptin secretion
and lactate dehydrogenase release to the medium (not shown) revealed
signs of tissue damage along the incubation period. In the 24-h
periods, leptin secretion significantly increased until 48 h in
samples from both sexes. Afterward, at either 72 or 96 h, the
secretion was well preserved, but without significant increase,
indicating that the in vitro model reached a plateau state
of leptin secretion; for this reason the integrated secretion was
analyzed in 48-h periods, as previously described (45). The leptin
secretion in the 48-h period was significantly higher in women than in
men (P < 0.05). Analyzed as the integrated secretion
(AUC as nanograms of leptin per g tissue/48 h; see Fig. 4), leptin
secretion by female samples (n = 33; 3904 ± 347) was
significantly higher (P < 0.05) than that by male
samples (n = 34; 2940 ± 323).
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). In contrast, dexamethasone induced a
clear-cut stimulation of leptin secretion in adipose tissue samples
from female donors. In fact, when the 24-h incubation periods were
considered, a significant difference (P < 0.01)
vs. the male samples was observed during the 48-h period
(Fig. 2A), although the stimulatory action of the glucocorticoid was
not evident at longer periods. When the integrated leptin secretion was
analyzed (Fig. 4
), the effect of dexamethasone was significantly
(P < 0.05) higher in female samples (n = 12;
5848 ± 624) than in control samples and was higher
(P < 0.05) than the secretion in samples from male
donors (n = 6; 3353 ± 741).
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). Analyzed as the integrated secretion (Fig. 4),
forskolin suppressed leptin secretion in both female samples (n =
3; 2677 ± 842) and adipose tissue from male donors (n = 5;
1421 ± 474); both were significantly lower (P <
0.05) than that in controls.
When adipose tissue fragments from nine women and six men were
incubated for 96 h in the presence of estradiol (1 µmol/L), the
spontaneous rate of leptin secretion in samples from male donors was
again unaffected, whereas a clear increase was observed in the
incubation medium of female adipose tissue (Fig. 3A). The stimulatory action of estradiol
was evident during the 48-h period (P < 0.05) and
vanished thereafter despite the continuous presence of the steroid in
the medium. Analyzed as the AUC (Fig. 4),
estradiol in female adipocytes induced a large leptin secretion (n
= 9; 5698 ± 688) that was significantly higher (P
< 0.05) than in the control studies. On the contrary,
estradiol-mediated leptin secretion in male samples (n = 6;
3373 ± 444) was not different from the spontaneous release in
male samples and was significantly (P < 0.05) lower
than that in estradiol-treated female samples. As shown in Fig. 3B and 3C, incubation of adipose tissue fragments with estrone (1 µmol/L) or
progesterone (1 µmol/L) over the 96-h period did not modify the rate
of spontaneous leptin secretion in any gender. The AUC of leptin
secretion after estrone administration was 3273 ± 307 for women
(n = 8) and 2871 ± 984 for men (n = 6), and after the
addition of progesterone to the medium, the AUCs were 3830 ± 602
(n = 6) and 2510 ± 545 (n = 5) for women and men,
respectively (Fig. 4).
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). No correlation was found between the
AUC of leptin secreted by each individual and relevant auxological
parameters, such as weight, BMI, or age.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Organ culture has been widely used in endocrinology (46); it has the advantage of convenience, lack of enzymatic damage to cellular structures, and most relevant, the fact that small adipose cells are not lost as is the case when adipose cells are isolated after enzymatic dispersion (47). In addition, the contribution of abdominal omental adipose tissue to the amount of circulating leptin is qualitatively similar to that of subcutaneous fat (7, 8, 48); the former is easier to prepare and manipulate due to its relative lack of fibrous and nonadipose components. In the present work, in vitro leptin secretion was followed for up to 96 h with no morphological or biochemical signs of tissue damage and well preserved leptin secretion; this timing was maintained in all studies. The good response to glucocorticoids and the inhibition by forskolin further supported the functionality of the assay. As the exponential part of leptin secretion was observed in both spontaneous and stimulated leptin secretion to occur in the first 48 h of incubation, the integrated secretion in that period was used for comparative purposes (44, 45). In agreement with previous reports (49), no gender differences in adipocyte size or number were detected histologically. Moreover, neither the BMI of the donors, the adipocyte number, nor the cross-sectional area correlated with the integrated secretion of leptin, because patients with obesity were excluded as tissue donors. While we were preparing this manuscript, another report using adipose tissue organ culture was published (31).
When spontaneous leptin secretion was assessed in nonstimulated samples, it was observed that adipose tissue from female donors secreted significantly higher amounts of leptin than that from men. This finding is the first observation indicating that two pieces of adipose tissue with identical weight release different amounts of leptin dependent on the donor’s sex, and this may be the biological basis for the observed in vivo gender differences in serum leptin concentrations. There is as yet no explanation for these gender-based variations in leptin release, which have been observed in vivo from the earliest periods of infancy (19). The finding that at birth, the leptin concentrations in umbilical cord blood from girls is double than that in boys strongly suggests that the gender-based differences are established during the intrauterine period. The observed differences may be explained by a different sex-based codification of the secretory pattern for male- or female-derived adipocytes or, alternatively, by differences in the hormonal milieu of the fetus that determine a different "adipocyte sex" in terms of leptin regulation.
It has been reported that an increase in the intraadipocyte levels of cAMP led to a suppression of leptin secretion (27, 32). In the present work it was also shown that in human adipocytes the activation of adenylate cyclase by forskolin inhibited leptin secretion, with no sex-based differences. The synthetic glucocorticoid dexamethasone elicits a clear-cut stimulation of leptin release both in vivo and in vitro in humans and experimental animals (24, 25, 26, 27, 29, 50, 51). In the present work, dexamethasone stimulated leptin release with a time lag of 24 h before becoming evident at 48 h, and it was not observed afterwards despite the continuous presence of the glucocorticoid in the incubation medium. This time course parallels that observed in vivo in obese subjects (52). Interestingly enough, this is the first time that a gender-based difference in the action of glucocorticoids has been reported. In fact, in vitro dexamethasone clearly increased leptin release in adipose tissue samples from female donors, whereas it was completely devoid of action in samples from men. There is no possibility to ascertain whether this gender-based difference is also operative in man in vivo due to the lack of gender-based analysis in other published reports where leptin results from both sexes were pooled (25, 52). There is no clear explanation for the sex-mediated differences in the glucocorticoid action on the adipocytes. A glucocorticoid response element has been reported in the promoter region of the genomic structure of the human obese gene (53), but women to men differences in genomic organization are unlikely.
Estrogen is involved in the regulation of circulating leptin levels in man (39, 54). Moreover, it has been reported in vitro that adipose tissue from rats increases leptin release after the administration of estradiol to the incubation medium (29, 55). Although the mechanism of estradiol action is not fully known, the obese gene has a consensus sequence of the estrogen-responsive element (56, 57) in its promoter region, and high affinity estrogen binding macromolecules are present in the cytosolic fraction of various adipose tissues (58). In the present work, estradiol was a powerful stimulator of leptin release, similar to dexamethasone, a fact that may explain the higher leptin concentration in women’s blood as well as the leptin changes throughout the menstrual cycle (54). Again, the most unexpected finding was the absence of leptin response after estradiol challenge in adipose tissue obtained from men, repeating the finding discussed above for dexamethasone. There is no clear explanation for the absence of reactivity to such stimuli, as adipocytes from men should have some receptors for estradiol and a similar amount of receptors for glucocorticoids compared with women’s fat cells. However, not all circulating steroids play a role in leptin secretion. Progesterone, a sex-related steroid hormone, and estrone, a weak estrogenic steroid, were devoid of stimulatory or inhibitory action on leptin secretion in adipose tissue from both women and men. This ruled out any nonspecific effect in the observed stimulatory actions of both dexamethasone and estradiol.
The clear-cut leptin responses to different stimuli such as dexamethasone and estradiol observed in adipose tissue from women vividly contrasts with the absent or scarce leptin secretion in tissues from men. This fact may be the cellular basis for the reported gender differences in serum leptin values and suggests a sex-dependent regulation of the leptin secretion in human adipocytes. It may also explain why in men the correlation between leptin values and fat mass is stronger than that in women, in whom for any amount of adipose tissue a wide dispersion of leptin values is observed. In any case, leptin levels more accurately reflect the net fat mass in men than in women, in whom they are reflecting both the fat mass and the responsiveness of these tissues to some changing hormonal components of blood. These points should be considered for further interpretations of the physiology of leptin.
In conclusion, leptin secretion from adipose tissue in vitro 1) is significantly higher in samples from women than in samples from men; 2) is stimulated by dexamethasone and estradiol in women, but not in men; 3) is not modified by progesterone or estrone in both sexes; and 4) is inhibited by forskolin in both sexes. The adipose tissue organ culture may be a useful tool for further understanding the regulation of leptin secretion in man.
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Acknowledgments |
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Footnotes |
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Received December 8, 1997.
Revised February 11, 1998.
Accepted February 23, 1998.
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, P < 0.05
vs. women’s sample values with the same treatment.
