René Pecquery

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.

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.

Effects of adiponectin on human trophoblast invasion

Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory, anti-atherogenic, and anti-proliferative effects. The expression of specific adiponectin receptors in the placenta and in the endometrium suggests a role for this cytokine in placental development, but this role has not yet been elucidated. The invasion of trophoblast cells during the first trimester of pregnancy being crucial to placentation process, we have studied adiponectin effects on human trophoblast invasive capacities. We found that adiponectin stimulated human trophoblast cell migration in HTR-8/SVneo cells in a dose-independent manner. In addition, adiponectin also significantly enhanced invasion of HTR-8/SVneo cells and of human extravillous trophoblast from first trimester placenta. These pro-invasive effects of adiponectin in human trophoblasts seem to be mediated in part via increased matrix metalloproteinases (MMP2 and MMP9) activities and via repression of TIMP2 mRNA expression. Our results suggest that adiponectin could be a positive regulator of the early invasion process by modulating the MMP/TIMP balance. Moreover, these results provide an insight into the role of adiponectin in pathological conditions characterized by insufficient or excessive trophoblast invasion.

Increased adenylate cyclase catalytic activity explains how estrogens in vivo promote lipolytic activity in rat white fat cells

Estradiol administration (5 micrograms per day x 4 days) to ovariectomized rats resulted in a 60-70% increase in the maximal lipolytic response of their white adipocytes to isoproterenol, epinephrine, IBMX and forskolin. These altered lipolytic responses were accompanied by parallel changes in the intracellular cyclic AMP levels found in response to 1 mM IBMX alone (+ 106%) or combined with submaximal concentrations of isoproterenol (+205%), epinephrine (+190%) and forskolin (235%). Studies of the adenylate cyclase activity revealed an overall increase in the stimulatory responsiveness of the enzyme (+150 to +200%) after the estradiol-treatment, regardless of the stimulatory agents tested (GTP, GppNHp, fluoride, isoproterenol, ACTH, forskolin). Finally, the finding of a 2-fold enhancement of the Mn2+ (+/- GDP beta S)-stimulated adenylate cyclase activity after the estradiol-treatment strongly suggests that increased activity of the catalytic subunit of this enzyme is the likely mechanism whereby estrogens promote lipolysis in rat fat cells.

Direct biochemical evidence for the existence of α-adrenergic receptors in hamster white adipocyte membranes

In fat cells, the first evidence confirming the coexistence of functionally opposed (Yand P-adrenergic receptors as regulatory subunits of adenylate cyclase [ 1] was obtained with human adipocytes [2]. However, it was rapidly felt that this concept could not be extended to the fat cells of all species studied and particularly to the commonly used rat fat cells [3]. Recent studies have shown that hamster white fat cells have a peculiar behaviour which set them apart from rat adipocytes [4]. Indeed, experiments testing the influence of catecholamine and phentolamine on CAMP metabolism provided some evidence for the existence of a-adrenergic sensitivity in these cells, suggesting that hamster adipocytes may be an appropriate model to study some aspects of the hormonal control of human fat cells [4] ; To date, however, no attempt has been made to directly identify the cr-adrenergic receptors in hamster white fat cells. Recently, methods measuring the binding of the aadrenergic antagonist [3 H] dihydroergocryptine [5] have been successfully used for the direct identification of a-adrenergic receptors in different tissues [6-g] and cells [9]. Using these binding techniques, we now report, in hamster white adipocyte, the identification of [3H] dihydroergocryptine binding sites which have the characteristics expected of a-adrenergic receptors and which appear therefore to represent the

Heterogeneity and subcellular localization of hamster adipocyte α-adrenergic receptors: Evidence of α1-and α2-subtypes

Hamster white fat cells are known to display, like the human adipocytes [ 11, a mixed (Yand fl-adrenergic sensitivity [2,3], which set these cells apart from the more frequently used and almost exclusively P-sensitive rat white adipocytes [4]. Methods measuring the binding of the labeled antagonist [3H]dihydroergocryptine have been successfully used for the direct identification of cr-adrenergic receptors in different tissues [5--g]. Applying these binding techniques to a ‘crude’ membrane fraction prepared from hamster adipocytes, we have identified and characterized specific binding sites for [3H]DHEC in this preparation [9]. Although most of the characteristics expected of true physiological a-adrenergic receptors were fulfilled by these binding sites [9], some of their properties suggested that these sites may be heterogeneous or that only a part of them may represent the true a-receptor sites. This has led us to reexamine in detail some of the properties of these [3H]DHEC binding sites. We report here the subcellular localization of these sites, the results of competition experiments using two highly selective atand olz-adrenergic antagonists, prazosin and yohimbine [lo111 and finally binding data obtained with membranes treated under conditions shown to induce an irreversible block of the fat cell physiological a-responsiveness. We show that [3H]DHEC binding sites are almost exclusively

Direct binding studies do not support the existence of true α-adreno-receptors in rat white fat cells

Summary By direct binding studies using the mixed α 1 -and α 2 -antagonist 3 H-dihydroergocryptine ( 3 H-DHEC) and the α 1 antagonist 3 H-prazosin as ligands, we have attempted to provide evidence for the existence of α 1 -adrenergic receptors in rat adipocyte membranes. Specific binding of both ligands is rapid, is reversible, is of high affinity (K D for 3 H-DHEC and 3 H-prazosin : 17.4 ± 2.4 and 12.1 ± 2.7 nM respectively), is saturable (Bmax for 3 H-DHEC and 3 H-prazosin : 222 ± 15 and 228 ± 37 fmol/mg protein, respectively) and is consistent with binding to a single class of binding sites, suggesting that both ligands label the same sites. Displacement of 3 H-DHEC by different adrenergically active drugs showed the following order of potency : prazosin > (-)-isoproterenol > (-)-epinephrine > (+)-epinephrine > yohimbine. These data as well as the inability of (-)-norepinephrine and methoxamine (anα 1 -agonist) to displace 3 H-DHEC indicate that these 3 H-DHEC binding sites are not true α 1 -adrenergic receptors and therefore render doubtful the existence of such receptors in rat white adipocytes.

Androgen receptors in cultured rat adipose precursor cells during proliferation and differentiation: regional specificities and regulation by testosterone

Different studies suggest that sex hormones affect adipose tissue metabolism and deposition. To investigate the possibility that androgens may play a role in adipose tissue development, we have studied androgen receptors (AR) in rat adipose precursor cells from two different anatomical fat deposits, one deep intraabdominal (epididymal) and one subcutaneous (inguinal) during the proliferation and differentiation processes. AR were quantified by [3H]R1881 specific binding in whole cells and the nuclear fraction and were localized by immunocytofluorimetry in both the cytosol and the nucleus. During the proliferative phase, total AR level decreased from D3 to D6. At confluence (D5), AR were higher in epididymal (64±4 fmol/mg protein) than in subcutaneous (33±3 fmoles/mg proteins) preadipocytes and were up-regulated by testosterone but not by 5α-dihydrotestosterone or by 17β-estradiol. At differentiation (D10-11), nuclear AR decreased by 50% in both precursor fat cell populations when compared to the confluent state (D5) and AR were no more up-regulated but rather down-regulated by testosterone. Because AR are present in preadipocytes and are differently regulated by testosterone depending on the stage of proliferation and differentiation, this study suggests that testosterone may play a role in the control of the adipogenic process.

Regulation of white adipocyte guanine nucleotide binding proteins Gsα and Giα1–2 by testosterone in vivo: Influence of regional fat distribution

Abstract The influence of the androgenic status on the steady-state amounts of Gi α 1–2 and Gsα subunits was compared in hamster fat cell membranes from the femoral subcutaneous (FSC) and epididymal (EP) adipose tissues, using immunoblotting experiments. In sham-operated hamsters, Gi α 1–2 and Gsα steady-state amounts found in FSC fat cells were 38% and 40% reduced, respectively, as compared to EP adipocytes. In EP fat cells, castration induced a down-regulation of both Gi α 1–2 (−39%) and Gsα (−33%), whereas testosterone replacement restored Gsα, but not Gi α 1–2 levels, to control values. In contrast, these G protein α-subunits were insensitive to the androgenic status in FSC fat cells. These data provide the first evidence that the androgenic status can modulate the expression of both the Gi α 1–2 and Gsα subunits of the fat cell adenylate cyclase regulatory Gi and Gs proteins and that this modulation depends on the anatomical origin of these cells.