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Service de Biochimie, INSERM CJF 94–02, Faculté de Médecine Paris-Ouest, Université Descartes (ParisV) Centre Hospitalier de Poissy, 78303 Poissy Cedex France
Address all correspondence and requests for reprints to: Yves Giudicelli, Service de Biochimie, Centre Hospitalier, 78303 Poissy Cedex, France.
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Abstract |
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Introduction |
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Leptin acts through different types of specific receptors that are members of the cytokine receptor family (6). Leptin receptors are widely distributed including in brain and many peripheral tissues, suggesting that leptin may provide quantitative information about fat stores to a wide range of tissues.
Various hormones and drugs have been reported to regulate the leptin mRNA and protein expression. Among them, insulin (7, 8, 9, 10, 11), glucocorticoids (10, 11, 12, 13, 14), and the thyroid hormone, triiodothyronine (T3) (15, 16) are up-regulators of leptin mRNA and protein expression, whereas ß3-adrenergic agonists (17, 18) and thiazolidinediones (19, 20) are down- regulators.
It is well established that sex steroids are involved in the site-specificities of adipose tissue metabolism (21, 22, 23, 24). A sexual dimorphism seems to apply for leptin production as well because serum leptin levels are higher in premenopausal women than in both men and postmenopausal women and remain still higher in postmenopausal women than in men (25, 26).
These observations raise the question as to whether sex steroid hormones could directly or indirectly intervene in the regulation of leptin secretion and/or production by adipose cells. Different recent studies have attempted to answer this question. However, owing to the various protocols used (in vivo or in vitro investigations in human or rat models, studies of either androgen or estrogen effects, measurements of ob mRNA expression and/or leptin secretion, blood leptin, etc.), the influence of sex steroids on leptin expression and/or secretion still remains unclear. Therefore, the aim of the present study was double: 1) to investigate in the rat model, the influence of androgenic and estrogenic status on the ob gene mRNA expression in vivo and, if any, 2) to determine whether estrogens and androgens can directly modulate in vitro the ob gene mRNA expression and leptin secretion in isolated adipocytes from various fat deposits.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods
Animals and experimental protocols. Procedures with
experimental animals are authorized and followed the guidelines of the
Ministère of Agriculture (France).
Sprague Dawley rats were kept under controlled lighting conditions (light, 0600 h, dark, 0800 h) and constant temperature (21 C) with free access to water and food.
Male and female rats (150 g) were gonadectomized or sham-operated (controls) as previously described (27, 28) and were killed by decapitation 2 or 3 weeks later, respectively, after an 18 h fasting. At the time they were killed, their body weights were 230–280 g.
Isolated adipocytes and culture conditions. Mature adipocytes were isolated from sc, perirenal, and parametrial adipose tissues using the collagenase digestion procedure described by Rodbell (29) with slight modifications. Following decapitation, adipose tissues were immediately removed under sterile conditions. Each fat pad was finely chopped before being transferred into the digestion solution consisting of DMEM-Ham’s F12) (50:50), collagenase 0.5–1 mg/ml, (BSA) 2%, streptomycin, 0.1 mg/ml, and penicillin, 100 U/ml.
Digestion was allowed to proceed with vigorous shaking for 30 min at 37 C, after which the completed digest was filtered through a 250 µm sterile nylon meshcone. The resulting filtrate was centrifuged for 3 min at 28 x g, the infranatant withdrawn, and the floating adipocytes washed three times with DMEM-Ham’s/F12 containing BSA 1%, streptomycin 0.1 mg/ml, and penicillin 100 U/ml before being kept in culture.
Aliquots (1 ml) of the adipocyte suspension (1–2 x 106 cells/ml) were rapidly dispensed and incubated for 24 h at 37 C under 5% CO2 and 95% air atmosphere in 5 ml of DMEM-Ham’s/F12 medium containing BSA (1.5%), leupeptin (0.2 nM), PMSF (100 nM), vitamin E (4 mg/ml), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and, when indicated, the hormones and drugs tested. At the end of these incubations, adipose cells were immediately used for RNA extraction and the culture medium stored at -80 C until it was assayed for leptin.
RNA extraction. Total cellular RNA was extracted following the guanidium isothiocyanate procedure described by Chomczynski et al. (30). The yield and quality of the extracted RNA were assessed by the 260/280 nm optical density ratio and by electrophoresis under denaturing conditions on 2% agarose gel.
Northern blot analysis. Total RNA (10 µg) were separated on denaturating gels containing 1% agarose and 12.5% formaldehyde. RNAs were capillary transferred onto a Hybond N+ membrane in NaOH (0.05 N) during 3 h and cross-linked to this membrane (1 h at 80 C). Prehybridizations were carried out for 3 h at 68 C in Church solution (0.5 M sodium phosphate, 1 mM EDTA, and 7% SDS) (31). Hybridizations with [32P]-labeled complementary DNA (cDNA) probes labeled by random priming (2–5 x 108 dpm/µg) were performed overnight at 68 C. The hybridized membranes were then washed twice in 2 x SSC (30 mM Na3 citrate, 300 mM NaCl pH = 7) with 0.1% SDS at room temperature and one time at 45 C before being exposed to an x-ray film at -80 C. The band intensities were quantified by densitometry.
To verify the amounts of RNA loaded, parallel gels were run and stained with ethidium bromide (0.3 mg/ml) to visualize ribosomal RNAs (28S and 18S).
Specific rat ob and ribosomal acidic protein (RP) (32) probes were prepared by RT-PCR (33) from adipocyte total RNA. Probes were then extracted by the phenol/chloroforme extraction procedure.
RT-PCR. One-half microgram of total RNA was denatured for 10 min at 72 C and was reversed transcribed to cDNA by incubating with 10 µl RT reaction mixture containing: 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM of each dNTP, 62.5 mU RNA guard, 50 ng random hexamers and 100 U Superscript II reverse transcriptase. Incubation was performed at 42 C for 60 min, heated to 95 C for 5 min, and then quickly chilled on ice.
The PCR reaction mixture contained 2 µl cDNA, 0.2 mM of each dNTP, and the Taq polymerase buffer, which contained 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 7.5 pM of each primer and 1.5 U of Taq polymerase.
PCR conditions were a denaturation step at 95 C for 2 min followed by 28 cycles of 95 C, 1 min; 55 C, 1 min; 72 C, 1 min. PCR were performed with a Cyclone thermocycler (Integra Bioscience, Cergy, France). PCR products were analyzed on a 2% agarose gel in 90 mM Tris-borate, 2 mM EDTA buffer (TBE), pH 8, and visualized by staining with ethidium bromide and UV transillumination.
In this semiquantitative RT-PCR method, two different primers sets were used: one primer set used for the rat ob gene had the following sequences: sense 5'-GAC ACC AAA ACC CTC ATC AAG-3' and antisense 5'-ATG TCC TGC AGA GAG CCC TG-3'. With this primer set, PCR generated a 383-bp fragment. To further characterize the amplimer, the PCR product was cleaved with AvaII. To be sure that amplification of the ob gene was within the exponential range, different numbers of PCR (20–25-28–30-32–35 cycles) were run. Finally, 28 cycles of PCR amplification were used to detect the ob mRNA.
The second primer set was specific for the rat cyclophilin gene, which is an ubiquitously housekeeping gene and can thus be used as internal standard (34). Rat cyclophilin gene specific primers had the following sequences: sense 5'-GGG AAG GTG AAA GAA GGC AT-3' and antisense 5'-GAG AGC AGA GAT TAC AGG GT-3'. With this primer set, PCR generated a 290-bp fragment of the cyclophilin gene. In the same way, different numbers of PCR cycles (25–27-29 cycles) were run. Finally, 27 cycles of PCR amplification were found to be optimal for detection of the cyclophilin mRNA.
Controls without reverse transcriptase were systematically performed to detect any cDNA contamination.
Leptin assay. Leptin levels in blood and cell incubation medium were determined with a commercially available RIA kit specific for rat leptin (35) using a rat leptin antibody produced in guinea pig. Leptin concentrations were within the detection range of the kit i.e. 0.5–50 ng leptin/ml.
Statistical analysis. All values were expressed as means ± SEM of three to four different experiments, and statistical analysis were performed using unpaired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In normal female rat, ob
mRNA levels were higher in parametrial (1.76-fold) and perirenal
(2.25-fold) than in sc adipocytes. In normal male rat, however, ob mRNA
levels were not influenced by the origin of adipose tissue.
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As shown in Fig. 2, castration for 2
weeks induced a 2-fold increase in ob mRNA expression in deep adipose
tissue (perirenal) and inversely a 50% decrease in ob mRNA expression
in femoral sc adipose tissue. Ovariectomy for 3 weeks resulted in a
25% decrease in ob mRNA level only in adipocytes from deep
localization (perirenal) and in a slight but not significant decrease
in femoral sc adipocytes.
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Serum leptin levels were also compared in these animals. We found no difference between male and female rats, and between castrated or ovariectomized rats compared with the sham-operated animals, respectively (data not shown). These observations are consistent with other studies (36, 37) showing no influence of 2- to 4-week ovariectomy on rat serum leptin levels.
In vitro studies
For the following in vitro studies, ob mRNA expression
was measured by a semiquantitative RT-PCR method using lower amounts of
total RNA than for the Northern blot analysis. It must be noticed that
when applying this RT-PCR method to the RNA preparations issued from
the in vivo experiments described above, the results
obtained were identical to those described in Figs. 1
and 2 (data not
shown).
We have chosen to investigate the direct effects of androgens on leptin release and ob mRNA expression in adipocytes from normal female rats for the following reasons. First, in normal female rats plasma testosterone levels were extremely low (<0.1 ng/ml) and not different from the circulating leptin levels found in castrated rats. Second, in preliminary experiments, we did not observe any difference in the basal secretion of leptin in culture medium when deep and sc adipocytes from castrated male and normal female rats were compared. Third, we have also shown that androgen receptor numbers in adipocytes from the same anatomical origin were not influenced by sex in human (38) and in rats (unpublished data).
Deep (perirenal and parametrial) and superficial (femoral sc) adipose cells from female rats were maintained in culture and samples of culture medium were removed after 24 h for leptin secretion measurements. Adipocyte total RNAs were also extracted at the end of the 24 h incubation period and ob mRNA levels measured.
First, we studied in vitro the ob mRNA expression according to the anatomical origin of the fat cells. We confirmed our in vivo observations, i.e. a higher expression of ob gene in deep than in superficial fat pads (data not shown). Second, we examined the rate of leptin secretion as a function of the incubation time and found it linear throughout the incubation period tested (24 h) (data not shown).
We next examined the effect of dihydrotestosterone, a metabolite of testosterone that is not metabolized into estrogens, on ob expression in perirenal and femoral sc adipocytes from normal female rats. With different dihydrotestosterone concentrations (10-9, 10-8, and 10-7 M free hormone concentration) we observed a dose-dependent inhibition of ob mRNA expression (data not shown). Therefore, 10-8 M free concentration of dihydrotestosterone was selected for the following experiments.
As can be seen in Fig. 3A, exposure to
dihydrotestosterone (10-8 M) resulted in a 25
and 30% decrease in ob mRNA expression in perirenal and sc adipocytes
respectively. Under the same experimental conditions, a parallel but
not significant (-20%) decrease occurred in the amount of leptin
secreted into the culture medium (Table 1). The effects of dihydrotestosterone on
ob mRNA were prevented when the adipocytes were exposed to cyproterone
acetate (10-6 M), which is a potent androgen
receptor antagonist, or to the transcriptional inhibitor, actinomycin D
(5 µg/ml) (Fig. 3A). None of these compounds had an effect per
se on ob mRNA. It is worth noting that incubation with
dexamethasone (10-8 M) used as a positive
control resulted in a 4- and 5-fold increase in leptin secretion from
sc and perirenal adipocytes respectively. Furthermore, the amount of
released leptin observed under control conditions, were higher in
perirenal than in sc adipocytes (Table 1). These regional specificities
of leptin secretion were correlated with the regional differences
observed for ob gene expression (Fig. 1).
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, exposure to
17-ß estradiol (10-8 M final concentration
of free hormone) for 24 h led to a significant increase in ob mRNA
expression in sc (+40%), perirenal (+20%) and parametrial
adipose cells (+75%). Under the same conditions, the leptin released
into the culture medium was increased by 65%, 109%, 288% in
perirenal, parametrial and sc adipocytes, respectively (Table 2). Moreover, these effects of 17-ß
estradiol were dose dependent (10-9, 10-8,
and 10-7 M free hormone concentration) (not
shown) and were prevented when the adipocytes were exposed to 17-ß
estradiol and either ICI182780 (10-6
M) a selective estrogen receptor antagonist or the
transcriptional inhibitor actinomycin D (5 µg/ml) (Fig. 3B, Fig. 4). None of these compounds had an effect
per se on ob mRNA and leptin secretion. Finally, as
described in Table 1, the leptin secretion was higher in deep than in
sc adipocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Confirming previous reports (39, 40) in the rat, we have observed that the steady-state ob mRNA level, in female rat, is lower in sc than in deep abdominal adipose tissue. The same picture was observed in cultured rat adipocytes: ob mRNA levels and the amount of leptin secreted are lower with sc than with perirenal and parametrial adipocytes. These observations are completely opposed to those found in humans where several studies have demonstrated that ob mRNA level is lower in deep than in sc fat deposits (41, 42, 43).
From the present study, there are obvious regional differences in the regulation of ob gene expression by the androgenic status in vivo because castration resulted in either a decrease (-50%) or an increase (+92%) of the ob mRNA level depending on whether sc or perirenal adipocytes were tested. These discrepancies could be explained by the higher aromatase activity in sc than in perirenal adipocytes (44). Indeed, in sc fat deposit from control male rats, endogenous androgens are preferentially metabolized to estrogens, which seem to regulate positively ob mRNA level as will be discussed below. Thus, after androgen withdrawal, the positive effect of the androgens derived estrogens may disappear in sc adipocytes leading to a decrease level of ob mRNA, and at the same time, the potent negative effect of androgens in perirenal adipocytes could also disappear thus explaining the increase in ob mRNA expression.
However, no such site-related differences could be observed in our in vitro study where a 24-h exposure to the nonaromatizable metabolite of testosterone, dihydrotestosterone (10-8 M), slightly (25 to 30%) but significantly reduced the ob mRNA level in both the perirenal and sc adipocytes from normal female rats.
These effects of androgens seem to be mediated through the adipocyte androgen receptors (38, 45, 46) because cyproterone acetate, a potent antagonist of these receptors, prevents the negative influence of androgens on ob gene expression. Reduced transcription rate or decreased transcript stability may account for this modulation. The finding that actinomycin D completely prevents the negative effects of dihydrotestosterone in vitro suggests that androgens reduce ob gene expression through transcriptional mechanisms.
Under our experimental conditions, however, we have observed that the levels of ob mRNA in control cells declined by 50% between the experiment start point (time zero) and the end of incubation (24 h) (data not shown). Therefore, we cannot firmly state that the effects of androgens are solely related to transcriptional inhibition of the ob gene expression or to a decrease in the stability of the ob mRNA.
On the other hand, the nonsignificant reduction of leptin release presently observed after 24-h exposure to dihydrotestosterone could be explained by the time of exposure used as it is very short compared with another study (47) describing a negative in vitro effect of androgens on leptin secretion in differentiating human fat cells after exposure to androgens for up to 12 days. In hypogonadal men (48), clinical studies have demonstrated that at baseline, serum leptin levels were 3-fold higher than in normal men, and, after testosterone treatment serum leptin levels were completely normalized. In another recent report (49), it was demonstrated that testosterone administration in female to male transsexuals decreased median serum leptin levels. Moreover, it is now clear that men have significantly lower serum levels of leptin compared with premenopausal or postmenopausal women, even when leptin levels were corrected for differences in body composition or fat mass (37, 48, 50). These studies are consistent with our present study demonstrating a direct negative control of androgens on ob gene expression and leptin production and/or secretion in the rat.
Concerning the estrogenic status, our in vivo experiments showed that ovariectomy caused a slight decrease (-25%) in the ob mRNA level in perirenal adipocytes. These results are also consistent with the recent study of Shimizu et al. (37) who also reported reduced ob gene expression in sc and retroperitoneal adipose tissues of ovariectomized rats and its restoration to normal level by a substitutive estradiol treatment. However, this study also showed increased ob gene expression in mesenteric adipose tissue after ovariectomy, suggesting a site specificity in the way whereby the gonadal status influences ob mRNA expression. In addition, our in vitro experiments provided clear evidence that the ob mRNA is overexpressed when adipocytes from ovariectomized rats are incubated with estrogens whatever the anatomical origin of the cells. In parallel to ob mRNA overexpression, the amount of leptin secreted into the culture medium was also increased. Moreover, the effects of 17-ß estradiol on ob gene expression and leptin secretion were prevented by actinomycin D and the antiestrogen ICI182780. Because adipocytes express estrogen receptors (51, 52, 53), these findings suggest a direct transcriptional effect of estrogens on ob gene expression. Giving further weight to this suggestion are i) our observation that the ob gene promoter region includes the half-palindrome of the estrogen-responsive element (ERE) present as a functional ERE in the chicken ovalbumin gene promoter (54) and ii) one recent report, demonstrating the presence of several Sp1 responsive element in the promoter region of the ob gene (55). Thus, an estrogen-receptor (ER)-Sp1 association could represent an estrogen induced transactivation pathway that would not require direct ER binding to DNA (56). Like in the androgen studies, however, we also observed a 40–50% decrease in the steady-state ob mRNA level of control adipocytes during the 24-h incubation period. Therefore, it cannot be excluded that estrogens may also increase the ob mRNA stability. Whatsoever, our present in vivo and in vitro findings showing that estrogens regulate positively the ob gene expression, could contribute to explain why ovariectomized rats are hyperphagic and become obese (57). On the other hand, estrogen regulation of central leptin receptors should also be considered. In a recent study (58), administration of estrogens to ovariectomized rats was found to cause an increase in the leptin-RL/leptin-Rs ratio, which may enhance the hypothalamic sensitivity to leptin leading to a subsequent reduction in food intake and body weight. Thus it appears that estrogens do not solely increase the expression of ob gene in white adipocytes, the main source of leptin (our present study), but also influence the expression of the ob receptors in the hypothalamus, one of the most important targets of leptin.
In nonobese mice and humans, it has been shown that a large proportion of leptin circulates bound to several serum proteins that may facilitate the transport of leptin across the blood-brain-barrier to its hypothalamic site(s) of action (59). Another study reported higher cerebrospinal fluid/plasma leptin ratios in women than in men (60). Beside their effects on leptin expression and secretion, we actually do not know whether estrogens could also influence the turnover and/or the function of these leptin binding proteins and thus modulate further the biological effects of leptin.
Chien et al. (61) have demonstrated that serum leptin levels just like serum estrogen levels, increase with the gestational age during normal rat pregnancy and sharply decrease just before parturition. Leptin receptor mRNA levels in the uterus are also up-regulated during pregnancy, whereas they do not change in other tissues such as the kidney, liver, ovary, and lung. These observations suggest an important physiological role for leptin during pregnancy and more specifically in the regulation of energy expenditure. Taking into account the results of the present study, it can be reasonably postulated that the increase in estrogen production and secretion that occurs during pregnancy is at least in part responsible for the increase in leptin production and possibly for the leptin receptor mRNA up-regulation in uterus during pregnancy.
In conclusion, our study indicates that androgens have a negative effect and, conversely, estrogens a positive effect on leptin expression. These modulation effects are direct, mediated by the adipocyte androgen and estrogen nuclear receptors and affect the ob gene transcription. However, additional experiments will be necessary to further establish the mechanism of this regulation at the molecular level.
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Acknowledgments |
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Received April 17, 1998.
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References |
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2/ß-adrenergic
control of lipolysis in white fat cells: predominant
2-antilipolytic response in
testosterone-treated-castrated hamsters. Endocrinology 122:2590–2596[Abstract]
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