Yves Giudicelli

Leptin mediates a proliferative response in human MCF7 breast cancer cells.

Obesity is a risk factor of breast cancers. As leptin, a hormone mainly secreted by white adipocytes, elicits proliferative effects in some cell types, we tested the hypothesis that leptin could influence human breast cancer MCF-7 cell growth. Here we show that MCF-7 cells express leptin receptors and respond to human recombinant leptin by STAT3 and p42/p44 MAPkinase activations and by increased proliferation. These findings suggest that leptin could act in vivo as a paracrine/endocrine growth factor towards mammary epithelial cells thus contributing to explain why obesity is a risk factor of developing breast cancers.

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 Receptorγ 21

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).

Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids

Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.

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

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. Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates. It was shown that the NO synthase inhibitor, NG‐monomethyl L‐arginine (L‐NMMA), but not the biologically inactive enantiomer NG‐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. Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro. 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. 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.

Rapid Nongenomic E2 Effects on p42/p44 MAPK, Activator Protein-1, and cAMP Response Element Binding Protein in Rat White Adipocytes

In some tissues, rapid effects of estrogens have been described at the plasma membrane level including activation of the MAPK activity. In rat adipocytes, the present study demonstrates that physiological concentrations (0.1-10 nM) of E2 rapidly activate the p42/p44 MAPK. This effect was blocked by the pure estrogen antagonist, ICI 182 780, and appeared specific for E2 because 17alpha-E2, T, and progesterone failed to change the MAPK activity. Pertussis toxin; PP2, a selective inhibitor of Src family kinase; and wortmannin all reduced the magnitude of MAPK activation by E2 suggesting involvement of the Gi-protein/Src family kinase/PI3K pathway. Classical PKCs and MAPK kinase were also involved in MAPK activation by E2. Interestingly, this activation was observed in late but not early differentiated rat preadipocytes, and the immunoreactive ER(alpha) protein was detected only in adipocyte membrane, suggesting that the adipocyte membrane structure is required for the nongenomic effect of E2. Moreover, E2 induced a rapid nuclear translocation of MAPK together with a fast MAPK- dependent activation of cAMP response element binding protein leading to a transcriptional activation of cAMP response element binding protein-responsive genes and reported plasmids. However, the E2 increase in adipocyte activator protein-1 DNA binding does not seem to be fully explained by the E2 activation of the MAPK pathway. This study provides clear evidence for an additional nongenomic mechanism whereby estrogens may exert their control on adipose tissue metabolism.

Modulation of White Adipose Tissue Lipolysis by Nitric Oxide

In isolated adipocytes, the nitrosothiolsS-nitroso-N-acetyl-penicillamine (SNAP) andS-nitrosoglutathione stimulate basal lipolysis, whereas the nitric oxide (NO ⋅ ) donor 1-propamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA-NONOate) or NO gas have no effect. The increase in basal lipolysis due to nitrosothiols was prevented by dithiothreitol but not by a guanylate cyclase inhibitor. In addition the cyclic GMP-inhibited low K m , cyclic AMP phosphodiesterase activity was inhibited by SNAP suggesting that SNAP acting as NO+ donor increases basal lipolysis through a S-nitrosylation mediated inhibition of phosphodiesterase. Contrasting with these findings, SNAP reduced both isoproterenol-stimulated lipolysis and cyclic AMP production, whereas it failed to modify forskolin-, dibutyryl cyclic AMP-, or isobutylmethylxanthine-stimulated lipolysis, suggesting that SNAP interferes with the β-adrenergic signal transduction pathway upstream the adenylate cyclase. In contrast with SNAP, PAPA-NONOate or NO gas inhibited stimulated lipolysis whatever the stimulating agents used without altering cyclic AMP production. Moreover PAPA-NONOate slightly reduces (30%) the hormone-sensitive lipase (HSL) activity indicating that stimulated lipolysis inhibition by NO ⋅ is linked to both inhibition of the HSL activity and the cyclic AMP-dependent activation of HSL. These data suggest that NO ⋅ or related redox species like NO+/NO− are potential regulators of lipolysis through distinct mechanisms.

Direct in vitro effects of androgens and estrogens on ob gene expression and leptin secretion in human adipose tissue

In the present study, we have explored, in vitro, the possibility that short exposure to androgens and estrogens for 24 h may directly influence leptin expression (ARNm and secretion) in sc adipose tissue from men and women. In men, only dihydrotestosterone at high concentration (100 nM) induced a reduction in leptin secretion and ob mRNA level. In women, 17β-estradiol (10–100 nM) increased ob mRNA expression (+180 to +500%) and leptin release (+75%). Moreover, in adipose tissue of women, the estrogen precursors testosterone (100 nM) and dehydroepiandrosterone (1 µM) also induced an increase in leptin secretion (+84 and +96%, respectively), an effect that was prevented by the aromatase inhibitor letrozole. Finally, the stimulatory effect of 17β-estradiol observed in women was antagonized by the antiestrogen ICI182780. Altogether, these results suggest that the sexual dimorphism of leptinemia in humans is mainly owing to the estrogen receptor-dependent stimulation of leptin expression in adipose tissue by estrogens and estrogen precursors in women.

Sex steroids and leptin regulate 11β-hydroxysteroid dehydrogenase I and P450 aromatase expressions in human preadipocytes: Sex specificities

Adipose tissue is an important site of steroid hormone biosynthesis, as type I 11beta-hydroxysteroid dehydrogenase (HSD1), the enzyme responsible for the conversion of cortisone into cortisol and the P450 aromatase, the enzyme catalysing androgens aromatization into estrogens, are both expressed in human adipose tissue. In the present report, we have investigated the possibility that sex steroids and leptin could regulate these two enzymes in cultured preadipocytes from men and women intra-abdominal fat depots. In women preadipocytes, human recombinant leptin down-regulates HSD1 mRNA expression (-58%) and P450 aromatase activity (-26%). Conversely, leptin up-regulates the HSD1 (2.4-fold) and the P450 aromatase (1.6-fold) mRNA expression in men preadipocytes. In women preadipocytes, 17beta-estradiol strongly stimulates HSD1 mRNA expression (10-fold) and, in contrast, decreases by half the P450 aromatase expression. In men, 17beta-estradiol has no influence on HSD1 expression but up-regulates P450 aromatase mRNA expression (2.4-fold). Finally, androgens increase by a factor of 2.5-5 the mRNA expression of both enzymes in men. These findings suggest that sex steroids and leptin either increase or decrease local cortisol and estrogens productions in men or in women preadipocytes, respectively. They also indicate that steroid metabolism in adipose tissue is controlled by a coordinated regulation of P450 aromatase and HSD1 expressions. Finally, the important sex-specific differences described herein may also contribute to explain the sexual dimorphism of body fat distribution in humans.

Metabolic pathways involved in the oxidation of isopropanol into acetone by the intact rat

Abstract Isopropanol administered in a large (6 g/kg, orally) as well as in a lower dose (1 g/kg, I.P.) is slowly oxidized into acetone by the intact rat. Using two inhibitors, 3 amino-1,2,4-triazole and pyrazole, investigations on the hepatic enzymatic system involved in the oxidation of isopropanol show that catalase does not play an important part in this pathway, contrary to alcohol dehydrogenase which is the major enzyme responsible for this oxidation. Although isopropanol oxidation is mainly catalysed in the liver through alcohol dehydrogenase, no alteration of the hepatic extramitochondrial redox state occurs after the administration of a large as well as of a lower dose of isopropanol. From these experiments it may be concluded that alterations of the liver NAD+/NADH ratio, which seem to play an important part in the ethanol induced fatty liver, are not involved in the isopropanol induced one.

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.