Effects of 17beta-estradiol on preadipocyte proliferation in human adipose tissue: Involvement of IGF1-R signaling.

Estrogens are known to stimulate the proliferation of human preadipocytes. However, the molecular mechanisms underlying the increased cell growth by these steroids are poorly understood. In the present study, we have demonstrated that the proliferative effect of 17beta-estradiol involves the induction of both cell cycle gene expressions, c-myc and cyclin D1. Moreover, the mitogenic effects of 17beta-estradiol are suppressed by the pure antagonist ICI 182780 suggesting that estradiol action is mediated by estrogen receptor (ER). We have also shown that 17beta-estradiol is able to inhibit human preadipocyte apoptosis capacity as reflected by DNA fragmentation experiments and the mRNA expression of the pro- and antiapoptotic genes. Finally, 17beta-estradiol significantly induces both mRNA and protein expression of RIGF1 in human preadipose cells via ER and thus reinforces the signaling pathway of the proliferative factor, IGF1. Taken together, these data reinforce the concept of cross-talk between IGF1- and ER-signaling pathways in preadipocytes and indicate that IGFI may be a critical regulator of estrogen-mediated preadipose growth.

[Screening for Down syndrome using first-trimester combined screening followed by second trimester ultrasound examination in an unselected population]. / Dépistage de la trisomie 21 par le test combiné du premier trimestre suivi par l'échographie du second trimestre en population générale.

BACKGROUND: Recent studies have reported the efficacy of first trimester combined screening for Down Syndrome based on maternal age, serum markers (human chorionic gonadotropin, pregnancy-associated plasma protein A), and ultrasound measurement of fetal nuchal translucency. However, those do not incorporate the value of the widely accepted routine 20-22 week anomaly scan. STUDY DESIGN: We carried out a multi-centre, interventional study in the unselected population of a single health authority in order to assess the performance of first trimester combined screening, followed by routine second trimester ultrasound examination and/or screening by maternal serum markers (free beta-hCG and alpha-fetoprotein measurement or total hCG, alpha-fetoprotein and unconjugated estriol measurement) when incidentally performed. Detection and screen positive rates were estimated using a correction method for non verified issues. A cost analysis was also performed. RESULTS: During the study period, 14,934 women were included. Fifty-one cases of Down Syndrome were observed, giving a prevalence of 3.4 per 1000 pregnancies. Of these, 46 were diagnosed through first (N=41) or second (N=5) trimester screening. Among the 5 screen-negative Down syndrome cases, all were diagnosed postnatally after an uneventful pregnancy. Detection and screen positive rates of first trimester combined screening were 79.6% and 2.7%, respectively. These features reached 89.7 and 4.2%, respectively when combined with second trimester ultrasound screening. The average cost of the full screening procedure was 108 euro (120 $) per woman and the cost per diagnosed Down syndrome pregnancy was 7,118 euro (7,909 $). CONCLUSION: Our findings suggest that one pragmatic interventional two-step approach using first-trimester combined screening followed by second trimester detailed ultrasound examination is a suitable and acceptable option for Down syndrome screening in pregnancy.

[Late paternity: spermatogenetic aspects]. / Paternité tardive: Aspects spermatiques et génétiques.

The effect of maternal age on the risk of meiotic abnormality is well documented. In contrast little is known about the effect of the paternal age. The question of the risk related to paternal age is raised because of the increased demand of Assisted Reproduction Techniques for older men. This review focuses on the alterations of male semen parameters, testis histology and genetic risks related to age. The motility, vitality and morphology of spermatozoa and semen volume are found decreasing with age. Histomorphometric studies reveal various alterations including a thickening of the basal membrane when spermatogenesis is arrested. The number of germinal and Sertoli cells decreases with increased age. Up to 95 years old, we could find subjects with complete spermatogenesis. Chromosomal analyses in different studies have provided controversial results. Our investigation on subjects aged from 29 to 102 showed that the rate of aneuploidy in the group of aged subjects with preserved spermatogenesis was not statistically different from the young control group. However the incidence of postmeiotic aneuploidy was increased when spermiogenesis had stopped. On the other hand from epidemiological studies, autosomal dominant diseases are known to be associated with paternal age. However, in the case of achondroplasia and Apert syndrome, direct DNA sperm analysis did not reveal significant increase in the mutation frequency with paternal age.

Evidence for functional estrogen receptors alpha and beta in human adipose cells: regional specificities and regulation by estrogens.

Adipocytes are estrogen-responsive cells, but the quantitative expression and transcriptional regulation of the estrogen receptors (ER-alpha and ER-beta) in human adipocytes and their precursor cells are unclear. Using real-time quantitative PCR, we have demonstrated that both ER-alpha and ER-beta mRNA are expressed in human mature adipocytes with a large predominance of ER-alpha mRNA. Moreover, ER-alpha mRNA is identically expressed whatever the anatomic origin (intraabdominal and subcutaneous) of the adipocytes and the gender. ER-beta mRNA levels are higher in women compared with men, without regional differences. 17beta-Estradiol in vitro upregulates expression of both ER-alpha and ER-beta mRNA in subcutaneous adipocytes from women but only the ER-alpha mRNA in subcutaneous and intra-abdominal adipocytes from men. In preadipocytes, only the ER-alpha subtype was present. In the latter cells, estrogens in vitro had no influence on ER-alpha expression (mRNA and protein). The present study also shows that estrogens in vitro increase the AP-1, SP-1, and estrogen response element DNA binding activities in differentiated but not in confluent preadipocytes, suggesting that ER become functional during the course of adipogenesis. On the whole, these data are consistent with a predominant role of the ER-alpha subtype in mediating the effects of estrogens on human adipose tissue development and metabolism.

Down's syndrome screening with nuchal translucency at 12(+0)-14(+0) weeks and maternal serum markers at 14(+1)-17(+0) weeks: a prospective study.

BACKGROUND: Sonographic and biochemical methods for Down's syndrome screening have developed simultaneously, but independently. As a consequence, the rate of invasive procedures for fetal karyotyping has dramatically increased and become an important public health issue which needs to be controlled. One approach is to combine sonographic and biochemical results into a single risk assessment. METHODS: In a multicentre interventional study, nuchal translucency (NT) was measured between 12(+0) and 14(+0) weeks of gestation. Maternal serum markers (MSM) were measured between 14(+1) and 17(+0) weeks of gestation. Karyotyping was advised when: (i) NT was > or =3 mm; or (ii) the MSM-related risk was > or =1 in 250 at term. Karyotyping was delayed until after a maternal blood sample had been taken. NT and MSM were expressed as multiples of the medians (MoMs), and risks were calculated and tailored to the study population. A combined risk for NT and MSM was estimated retrospectively. Costs per case diagnosed, and the cost per case averted were calculated for the three screening strategies. RESULTS: A total of 9444 women was screened. Twenty-one fetuses (0.22%) had Down's syndrome, whilst 326 women (3.4%) were lost to follow-up. Among 9118 women followed up, 5506 had both NT and MSM, 821 had only NT, and 2791 had only MSM. Median maternal age was 30.5 years. False-positive rates for NT, MSM and NT combined with MSM were 3.0, 5.8 and 0.23% respectively. The false-positive rate generated by a sequential two-stage screening was 8.6%. Detection rates of Down's syndrome were 62 and 55% for NT and MSM respectively. Seven cases with Down's syndrome (35%) had raised NT and MSM, and 17 (81%) had either raised NT, MSM, or both. For a 5% false-positive rate, detection rates were 55 and 80% for NT alone and for combined NT and MSM respectively. Ultrasound alone appears to be more cost-effective ( pound50 per case diagnosed) than both tests ( pound61 per case diagnosed). CONCLUSIONS: The study results suggest a 25% increase in the detection rate of Down's syndrome using a combination of NT measurement at 12(+0)-14(+0) weeks and MSM at 14(+1)-17(+0) weeks for a 5% false-positive rate, with modest increase in cost.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways.

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

Differences in type II, IV, V and VI adenylyl cyclase isoform expression between rat preadipocytes and adipocytes.

Adenylyl cyclase catalytic activity is low in preadipocyte membranes when compared to adipocytes. Under conditions promoting inhibition of adipocyte adenylyl cyclase activity by Gpp(NH)p, a stable GTP analog, a paradoxical increase in preadipocyte adenylyl cyclase activity was obtained. In order to explain this contradiction, expression of types II, IV, V and VI adenylyl cyclase isoforms was compared in adipocytes and undifferentiated preadipocytes both by western blots and by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Type II, IV, V and VI mRNAs and proteins were present in both adipocytes and preadipocytes. However, in undifferentiated preadipocytes, expression of type II mRNA and protein were significantly higher whereas expression of type IV, V and VI adenylyl cyclase mRNAs and proteins were significantly weaker than in adipocytes. In late differentiated preadipocytes, the adenylyl cyclase subtype mRNA expression pattern was intermediary between the undifferentiated and the full differentiation states except for type IV which remained weakly expressed. Moreover, one of the representative regulators of G-protein signaling (RGS protein), RGS4, was less expressed in undifferentiated preadipocyte membranes and cytosol extracts, which contrasts with adipocytes where RGS4 is clearly expressed. Thus, the preferential expression of type II adenylyl cyclase (G(betagamma) subunit-stimulated) in preadipocytes might explain why Gpp(NH)p elicits stimulation of adenylyl cyclase under conditions designed to promote inhibition. Conversely, the preferential expression of type V and VI adenylyl cyclases and the slightly higher expression of type IV adenylyl cyclase in adipocytes could contribute to explain the elevated total catalytic activity observed in mature fat cells compared to their precursor cells.

An up-regulation of renal alpha(2)A-adrenoceptors is associated with resistance to salt-induced hypertension in Sabra rats.

This study investigates the incidence of high-salt diet in blood pressures, renal alpha(2)-adrenoceptor subtypes distribution, and gene expression in salt-sensitive (SBH) and salt-resistant (SBN) Sabra rats. Comparisons have been made between SBH and SBN rats submitted to a normal or a high-salt diet for 6 weeks. Only alpha(2)B-adrenoceptors are detected in kidneys of SBH rats, whatever the diet. In contrast, mRNA corresponding to alpha(2)A- and alpha(2)B-subtypes are found in this substrain. In these rats, high-salt diet increases blood pressures and up-regulates gene expression and density of only alpha(2)B-adrenoceptors. Inversely, alpha(2)A- and alpha(2)B-adrenoceptors and corresponding mRNA are found in kidneys of SBN rats. In these rats, a high-salt diet does not affect blood pressures but increases gene expression and densities of both alpha(2)A- and alpha(2)B-adrenoceptors. If the up-regulation of renal alpha(2)B-adrenoceptor subtypes is indicative of the hypertensive phenotype, the present study shows that this mechanism is also present in normotensive salt-resistant Sabra rats. In fact, the absence of alpha(2)A-adrenoceptors in SBH could be responsible for the lack of adequate receptor-mediated renal functions predisposing to salt-sensitivity and consequently the development of hypertension. Conversely, the presence of this receptor in SBN rats and its up-regulation could be protective change against the increase of alpha(2)B-adrenoceptors induced by the salt overload and could consequently be responsible for the resistance to salt-induced hypertension.

Site-specific control of rat preadipocyte adipose conversion by ovarian status: Possible involvement of CCAAT/enhancer-binding protein transcription factors.

The preadipocyte-adipocyte conversion process from two intraabdominal (parametrial and perirenal fat depots) is differently affected by ovarian status in the rat. We have tested the hypothesis that these site-specific alterations of adipogenesis might be related to changes in the expression of the transcription factors c-mycand CCAAT/enhancer binding proteins (C/EBPalpha, -beta, and -zeta) that regulate proliferation and differentiation. The increased proliferation rates observed in parametrial and perirenal preadipocytes after ovariectomy were not linked to variations in c-myc mRNA levels. Expression of the early marker of adipogenesis, lipoprotein lipase (LPL), remained insensitive to the ovarian status in early differentiated parametrial and perirenal preadipocytes. By contrast, LPL expression increased in early differentiated sc preadipocytes from ovariectomized rats, an effect that was completely reversed by in vivo estradiol and progesterone treatment. Expression of C/EBPbeta protein was unaffected by ovarian status whatever the anatomic origin of the preadipocytes. By contrast, the levels of p42 and p30 isoforms of C/EBPalpha were specifically decreased in parametrial preadipocytes, an alteration that was completely corrected by in vivo administration of estradiol and progesterone. C/EBPzeta, a dominant inhibitor of C/EBPalpha and -beta, exhibited a strong site-specific expression since C/ EBPzeta content was fivefold higher in sc preadipocytes than in deep intraabdominal cells whatever the ovarian status. Furthermore, ovariectomy selectively decreased C/EBPzeta levels in sc cells. In conclusion, our study suggests that some of the site-specific effects of ovariectomy on adipogenesis could involve, at least in part, altered expressions of C/EBPalpha and -zeta, both of which are important transcriptional regulators of fat cell differentiation and metabolism.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.

Androgen regulation and site specificity of angiotensinogen gene expression and secretion in rat adipocytes.

Adipose tissue is an important source of angiotensinogen (ATG), and hypertension is commonly associated with android obesity. Therefore, we tested the hypothesis that androgens may control ATG gene expression and secretion in rat fat cells. In intact male rats, ATG mRNA expression (Northern blot and co-reverse transcription-polymerase chain reaction analysis) and protein secretion were significantly higher in deep intra-abdominal (perirenal and epididymal) than in subcutaneous adipocytes. After castration, ATG mRNA was reduced almost 50% in the three fat deposits, with parallel changes in ATG protein secretion. Conversely, testosterone treatment fully restored the ATG mRNA decrease after castration, whatever the anatomical origin of the adipocytes. Finally, a 24-h in vitro exposure of perirenal fat cells or differentiated preadipocytes from castrated rats to testosterone or dihydrotestosterone (10 nM free hormone concentration) increased ATG mRNA expression by 50-100%, an effect that was prevented by the anti-androgen cyproterone acetate. These data, demonstrating both in vivo and in vitro androgen induction of ATG mRNA expression in rat adipocytes, add further weight to the hypothesis of a link between adipose tissue ATG production, androgens, and android obesity-related hypertension.

Endogenous nitric oxide is implicated in the regulation of lipolysis through antioxidant-related effect.

We studied the influence of nitric oxide (NO) endogenously produced by adipocytes in lipolysis regulation. Diphenyliodonium (DPI), a nitric oxide synthase (NOS) inhibitor, was found to completely suppress NO synthesis in intact adipocytes and was thus used in lipolysis experiments. DPI was found to decrease both basal and dibutyryl cAMP (DBcAMP)-stimulated lipolysis. Inhibition of DBcAMP-stimulated lipolysis by DPI was prevented by S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor. This antilipolytic effect of DPI was also prevented by two antioxidants, ascorbate or diethyldithiocarbamic acid (DDC). Preincubation of isolated adipocytes with DPI (30 min) before exposure to DBcAMP almost completely abolished the stimulated lipolysis. Addition of SNAP or antioxidant during DPI preincubation restored the lipolytic response to DBcAMP, whereas no preventive effects were observed when these compounds were added simultaneously to DBcAMP. Exposure of isolated adipocytes to an extracellular generating system of oxygen species (xanthine/xanthine oxidase) or to H(2)O(2) also resulted in an inhibition of the lipolytic response to DBcAMP. H(2)O(2) or DPI decreased cAMP-dependent protein kinase (PKA) activation. The DPI effect on PKA activity was prevented by SNAP, ascorbate, or DDC. These results provide clear evidence that 1) the DPI antilipolytic effect is related to adipocyte NOS inhibition leading to PKA alterations, and 2) endogenous NO is required for the cAMP lipolytic process through antioxidant-related effect.

Estradiol stimulation of c-fos and c-jun expressions and activator protein-1 deoxyribonucleic acid binding activity in rat white adipocyte.

In order to elucidate the molecular mechanisms whereby ovarian hormones, and particularly estrogens, modulate fat cell metabolism, we investigated the effects of estradiol administration on c-fos and c-jun expressions in fat cells from ovariectomized (OVX) rats. Estradiol treatment resulted in a rapid increase in c-fos and c-jun messenger RNA (mRNA) and protein levels (about 2-fold). These effects of estradiol on c-fos and c-jun mRNAs were blocked by actinomycin D but not by cycloheximide treatment, suggesting that estradiol modulates c-fos and c-jun transcription. Moreover, the estradiol-induction of both transcripts was partially suppressed by the estrogen-receptor antagonist ICI 182,780. In contrast, progesterone administration did not affect c-fos and c-jun mRNA levels indicating a hormonal specificity of estrogen action. However, an antagonism of estradiol-induction of both genes was observed after progesterone treatment. In addition, the estradiol-induced changes in c-fos and c-jun mRNA expressions could not be observed in castrated males suggesting a gender-specific effect of estradiol. Finally, in OVX rats, estradiol treatment stimulated the specific AP-1 DNA binding activity (about 5-fold) in adipocyte nuclear extracts as assessed by electrophoretic mobility shift assays. These results suggest that some of the estrogen effects in fat cells from female rats are mediated through induction of the AP-1 complex expression and consequently through modulation of the AP-1 dependent gene expression in adipocytes.

Hypertension in cafeteria-fed rats: alterations in renal alpha2-adrenoceptor subtypes.

Obesity is a major cause of human essential hypertension and there are clear evidences that abnormal kidney functions play a key role in obesity hypertension. Feeding rats a cafeteria diet has been extensively used as an experimental model to study obesity and energy balance expenditure. The present study investigated whether rats fed a cafeteria diet develop hypertension with alterations in renal alpha2-adrenoceptor subtype distribution. Weight gain induced by feeding rats a cafeteria diet during 8 weeks was associated with a marked increase in blood pressure. Insulin levels were higher in these hypertensive rats, leading to a decreased plasma glucose/insulin ratio. Based on radioligand-binding studies using [3H]-RX821002 and selective competitors, a raise in alpha2-adrenoceptor density that was solely due to an increased alpha2B-adrenoceptor subtype density was detected in the kidney of the cafeteria-fed rat. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) experiments showed an overexpression of the gene encoding the alpha2B-adrenoceptor subtype in these rats. On the other hand, despite a similar mRNA level, the alpha2A-adrenoceptor subtype was no more detectable by radioligand-binding studies in the kidney of the cafeteria-fed rat. In conclusion, cafeteria-fed rats are hypertensive, with renal alterations in alpha2-adrenoceptor distribution. These alterations, which are not related to genetic factors, may play a key role in the onset of hypertension.

Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2.

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17beta-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (x2) of 17beta-estradiol (0.01 microM) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 microM) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17beta-estradiol (0.01 microM), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17beta-estradiol significantly increased (x1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor gamma2 was up-regulated by 17beta-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 beta-estradiol (0.01 microM) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 microM) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor gamma2 expression (estrogens).

Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells.

The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular.

A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in vivo.

1. Possible nitric oxide (NO)-mediated effects on lipolysis were investigated in vivo in human subcutaneous adipose tissue using microdialysis, as well as in vitro on isolated fat cells of non-obese, healthy volunteers. NO donors were added to the ingoing dialysate solvents. 2. Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates. 3. It was shown that the NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA), but not the biologically inactive enantiomer N(G)-monomethyl D-arginine (D-NMMA), increased glycerol levels in the microdialysates without causing a change of local blood flow. In addition, L-NMMA increased glycerol levels in the microdialysate when local blood flow was stimulated with hydralazine. 4. Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro. 5. Expression of inducible nitric oxide synthase (iNOS) in human adipose tissue was shown by Western blot analysis. Biologically active NOS was demonstrated by measuring total enzymatic activity. 6. In conclusion, the data demonstrate that inhibition of NO release in subcutaneous adipose tissue results in an increased lipolysis in vivo. These effects, which were also observed in vitro, are independent of local blood flow changes. Furthermore, the demonstration of enzymatic NOS activity and the expression of inducible nitric oxide synthase (iNOS) in adipose tissue indicate that locally synthesized NO may play a role in the physiological control of lipolysis in human adipose tissue.

Modulation of rat preadipocyte adipose conversion by androgenic status: involvement of C/EBPs transcription factors.

Androgenic status affects rat preadipocyte adipose conversion from two deep intra-abdominal (epididymal and perirenal) fat depots differently. The aim of this study was to establish whether these site-specific alterations of adipogenesis are related to altered expressions of the transcriptional factors regulating proliferation and differentiation of preadipocytes, c-myc and CCAAT/enhancer binding proteins (C/EBPs: C/EBPalpha and beta). The increased proliferation of epididymal and perirenal preadipocytes from castrated rats was not linked to variations in c-myc mRNA and protein levels. The expression of the early marker of adipogenesis, lipoprotein lipase (LPL), was decreased by androgenic deprivation in epididymal cells but remained insensitive to the androgenic status in perirenal preadipocytes. In contrast, LPL expression increased in subcutaneous preadipocytes from castrated rats, an effect which was partly corrected by testosterone treatment. Expression of C/EBPbeta was unaffected by androgenic status whatever the anatomical origin of the preadipocytes. In contrast, the mRNA and protein levels of C/EBPalpha were greatly decreased by androgenic deprivation in epididymal cells, an alteration which could not be corrected by in vivo testosterone administration. Altogether these results demonstrated that in preadipocytes androgenic deprivation affects site-specifically the expression of LPL, an early marker of adipogenesis and of C/EBPalpha, a master regulator of adipogenesis. These observations contribute to an explanation of why castration induces defective adipose conversion in rat epididymal preadipocytes specifically.

In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones.

As a sexual dimorphism appears in plasma leptin levels, the aim of the present study was to investigate, in vivo and in vitro, the influence of sex steroid hormones on ob messenger RNA (mRNA) and leptin expressions in rat fat cells from various anatomical localizations. In male rats, castration resulted in a modulation of ob gene mRNA expression which was increased by 2-fold in perirenal and half-reduced in sc adipocytes. Moreover, in isolated fat cells from both perirenal and s.c. fat depots, ob gene mRNA expression was reduced by 20% after a 24-h in vitro exposure to dihydrotestosterone (10(-8) M). This effect of dihydrotestosterone on ob mRNA was prevented by exposure to the antiandrogen cyproterone acetate and also by actinomycin D. In contrast, leptin secretion from both perirenal and sc adipocytes was unchanged after 24 h exposure to dihydrotestosterone. In female rats, ovariectomy induced a 25% decrease in ob gene mRNA expression in perirenal fat cells. In vitro studies revealed that a 24-h exposure to 17-beta estradiol (10(-8) M) induced a 1.4-, 1.2-, and 1.75-fold increase in ob mRNA expression and a 3.8-, 1.65- and 2-fold increase in leptin secretion in sc, perirenal and parametrial adipocytes, respectively. Moreover, these effects were prevented by the antiestrogen ICI182780 and also by actinomycin D. Altogether, these results demonstrate that in rat adipocytes, estrogens, and androgens modulate ob gene expression at the mRNA level through sex steroid receptor-dependent transcriptional mechanisms.