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.

Catecholamine binding to the alpha-adrenergic receptors of hamster adipocytes. Evidence that guanine nucleotides regulate this binding to the alpha 2-receptor subtype.

The binding characteristics of the alpha-component of (-)-[3H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, equilibrium in 20 min at 25 degrees C. Dissociation of specific binding by 10 microM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [3H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (KD = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (KD = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands competed with [3H]norepinephrine binding in the following order of potency: (-)-norepinephrine greater than (-)-epinephrine much greater than (+)-norepinephrine greater than (-)-isoproterenol. Displacement by the selective alpha-adrenergic drugs prazosin, clonidine, and yohimbine yielded biphasic curves consistent with binding of [3H]norepinephrine to both alpha 1-(14-22%) and alpha 2-(78-86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [3H] dihydroergocryptine, it severely reduced the binding affinity of (-)-epinephrine, (-)-norepinephrine and the selective alpha 2-agonist, clonidine. The inhibitory effects of clonidine and of the alpha-component of (-)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [3H]norepinephrine and [3H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the alpha-inhibitory effect of norepinephrine on cyclic AMP production. These data led to conclude that [3H]norepinephrine can be successfully used to study the entire alpha-adrenergic receptor population of hamster fat cells and that the predominant alpha 2-receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.

Hormonal activation of the cGMP-inhibited low-Km cyclic AMP phosphodiesterase of rat adipocytes from different sites: influence of ovariectomy.

Hormonal activation of the cGMP-inhibited low Km cyclic AMP phosphodiesterase isoenzyme (cGI.PDE) by effectors, acting either through the cAMP-independent (insulin) or through cAMP-dependent (isoproterenol, forskolin ACTH and 8Br-cAMP) mechanisms, were compared in parametrial (PM) and femoral subcutaneous (SC) adipocytes from sham-operated (SHAM) and ovariectomized (OVX) rats. In SHAM rats, the basal cGI.PDE activity was 50% higher in PM than in SC adipocytes. In OVX rats, the cGi.PDE activatory responses to all the effectors tested remained unchanged in SC, but were completely suppressed in PM adipocytes. The mechanism underlying these defective cGI.PDE activatory responses to cAMP-dependent effectors observed in PM adipocytes after OVX seems to involve protein kinase A, since a decreased activation of cGI.PDE by protein kinase A was also found in these cells. Treatment of OVX rats with both estradiol and progesterone reversed the defective cAMP-dependent activation of cGI.PDE, but not the refractoriness of this isoenzyme to insulin activation. Taken together with previous observations from this laboratory on the fat cell adenylate cyclase system (Lacasa et al. (1991) Endocrinology 128, 747-753), these results: (a) demonstrate that the influence of the ovarian status on the key enzymes controlling cAMP metabolism in fat cells depends on the anatomical origin of these cells, and; (b) provide a biochemical explanation to the insensitivity of the SC adipocyte lipolytic system to ovarian hormones.

Characteristics of beta-adrenergic-agonist binding to rat adipocyte membranes. Evidence that (+/-)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte beta-adrenergic receptors.

The binding characteristics of the beta-adrenergic agonist (+/)-[3H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37 degrees C (second order rate constant K1 = 1.37 . 10(7) . M-1 . min-1). Dissociation of specific binding by 0.5 mM (-)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k2 of 0.106 . min-1 and 0.011 . min-1 . [3H]Hydroxybenzylisoproterenol binding was saturable (Bmax = 690 +/- 107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [3H]hydroxybenzylisoproterenol binding sites, one having high affinity (KD = 3.5 +/- 0.7 nM) but low binding capacity (10% of the total sites) and one having low affinity (KD = 101 +/- 20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [3H]hydroxybenzylisoproterenol binding with the following order of potency = (-)-propranolol greater than (-)-isoproterenol greater than (-)-norepinephrine approximately (-)-epinephrine greater than greater than (+)-isoproterenol = (+)-propranolol, which is consistent with binding to beta 1-adrenergic receptors. Competition curves of [3H]hydroxybenzylisoproterenol binding by the beta-agonist (-)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by beta-antagonist (-)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [3H]hydroxybenzylisoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (-)-isoproterenol, the number of [3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (-)-isoproterenol to stimulate adenylate cyclase correlated closely with the ability of (-)-isoproterenol to displace [3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the beta-antagonist [3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [3H]hydroxybenzylisoproterenol can be successfully used to label the beta-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [3H]dihydroalprenolol in these cells.

Is fatty liver induction a general feature of the administration of foreign sulfhydryl compounds?

The intraperitoneal administration of 2-mercaptoethanol or 2-mercaptoacetate (40 muM/100 g body weight) to the rat induces a fatty liver, a marked and early increase of free fatty acids and a decrease of triacylglycerol and phospholipids in the blood. These changes are accompanied by a decrease of the ketone body level and the beta-hydroxybutyrate/acetoacetate ratio in the liver. Under the same experimental conditions, however, administration of 2-mercaptopropionate fails to induce a fatty liver and does not modify the hepatic ketone body level or the blood triacylglycerol and free fatty acid levels. These results led us to conclude that fatty liver induction is not a general feature of foreign thiols, and suggest that increased peripheral fat mobilization as well as decreased hepatic lipoprotein synthesis and/or release are responsible for the 2-mercaptoethanol- and 2-mercaptoacetate-induced fatty liver.

Influence of trypsin on lipolysis in human fat cells. Comparison with rat adipocytes.

1. Trypsin-treated human and rat fat cells were obtained by digestion of adipose tissue with collagenase plus trypsin and their lipolytic response to insulin, catecholamines and dibutyryl cyclic AMP were compared with the lipolytic response of human and rat fat cells isolated with collagenase only. 2. In both human and rat fat cells, no significant modification occurred in the intracellular lactate dehydrogenase content and in the basal release of glycerol after trypsination. 3. In rat fat cells, trypsin abolished the antilipolytic effect of insulin but maintained a normal lipolytic response to epinephrine, norepinephrine and isoproterenol. 4. In human fat cells, on the contrary, trypsin failed to modify the antilipolytic effect of insulin, but markedly potentiated the lipolytic response to epinephrine, norepinephrine and isoproterenol. Trypsin also increased the rate of intracellular 3' :5' cyclic AMP accumulation in response to catecholamines. Under these conditions, however, trypsin-treated human fat cells had a normal reponse to the lipolytic agent dibutyryl cyclin AMP. 5. These data suggest that human fat cells differ from the rat ones by the existence in human adipocyte membranes of a trypsin-sensitive component which inhibits the catecholamine induced lipolytic process and which is different from the alpha receptors.

Characterization of the beta-adrenergic receptors of hamster white fat cells. Evidence against an important role for the alpha 2-receptor subtype in the adrenergic control of lipolysis.

The binding characteristics of the beta-adrenergic antagonist, [3H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity (Kd = 1.6 +/- 1.3 nM) but low capacity (32 +/- 17 fmol/mg membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both beta-antagonists and beta-agonists, competition at the low-affinity sites occurred only with beta-antagonists and was non-stereoselective. Thus, the beta-agonist (-)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity (Kd = 1.6 +/- 0.5 nM) binding sites with a binding capacity of 53 +/- 13 fmol/mg membrane protein (corresponding to 4000 +/- 980 sites per cell), and independent kinetic analysis provided a Kd value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a beta 1-receptor subtype, yielding Kd values in good agreement with the Kact and the Ki values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of beta-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [3H]dihydroalprenolol binding sites defined as the binding displaceable by (-)-isoproterenol represent the physiologically relevant beta-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (-)-norepinephrine showed that the inhibitory effect of the alpha 2-component of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the alpha 2-receptors in the adrenergic control of lipolysis in hamster white adipocytes.

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.

Rat preadipocyte adenylyl cyclase: influence of fat localization and androgenic status.

The influence of androgenic status on basal and stimulated cAMP production, adenylyl cyclase activities and immunoblot quantified GS alpha and Gi alpha 2 subunits of the adenylyl cyclase regulatory proteins were compared in confluent preadipocytes from subcutaneous (SC) and deep-intraabdominal (epididymal) fat deposits. Maximal cAMP response to isoproterenol was lower in SC than in epididymal preadipocytes. After castration, this site-specific difference was suppressed. cAMP response to 2-chloroadenosine, which was identical in the two types of preadipocytes, was decreased by castration in epididymal cells but not in SC cells. The catalytic activity of adenylyl cyclase and its maximal response to GTP were higher in epididymal than in SC preadipocytes. This response to GTP was decreased by castration in epididymal preadipocytes while it remained unchanged in SC preadipocytes. The catalytic activity of adenylyl cyclase was unchanged by androgenic status whatever the cell localization. Levels of GS alpha quantified by immunoblotting were not modified whatever the androgenic status and cell origin. Levels of Gi alpha 2 were not affected by the androgenic status as well, but were lower in SC than in epididymal cells. This study shows that components of the adenylyl cyclase system in preadipocytes are differently regulated by the androgenic status depending on the anatomical origin of the cells.

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.

Regulation of white adipocyte guanine nucleotide binding proteins Gs alpha and Gi alpha 1-2 by testosterone in vivo: influence of regional fat distribution.

The influence of the androgenic status on the steady-state amounts of Gi alpha 1-2 and Gs alpha 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 alpha 1-2 and Gs alpha 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 alpha 1-2 (-39%) and Gs alpha (-33%), whereas testosterone replacement restored Gs alpha, but not Gi alpha 1-2 levels, to control values. In contrast, these G protein alpha-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 alpha 1-2 and Gs alpha subunits of the fat cell adenylate cyclase regulatory Gi and Gs proteins and that this modulation depends on the anatomical origin of these cells.

G proteins in adipocytes and preadipocytes: characterization, subcellular distribution, and potential roles for Gi2 and/or Gi3 in the control of cell proliferation.

Guanosine triphosphate (GTP)-binding protein subunits were studied by immunoblot analysis in particulate fractions from mature adipocytes, confluent preadipocytes, and in vitro-differentiated preadipocytes. Mature adipocytes express Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha, Gq/11 alpha, G13 alpha and the long and short isoforms of Gs alpha, but no Gz alpha or G12 alpha. Confluent and differentiated preadipocytes differ in having a higher content of Gi alpha 3 and G13 alpha and expressing G12 alpha. In contrast, they lack Gi alpha 1, Go alpha, and the short from of Gs alpha. The G-protein alpha subunits Gi alpha 2, Gs alpha (long isoform), and Gq/11 alpha, and G-protein beta subunits were unchanged throughout the differentiation process. By immunoblot and indirect immunofluorescence studies on confluent preadipocytes, we showed that Gi alpha 2 is present in the endoplasmic reticulum and marginally in plasma membranes and nuclei. In contrast, antibodies to Gi alpha 3 stained the Golgi apparatus. The role of G proteins on preadipocyte proliferation was studied using Bordetella pertussis toxin. Exposure of growing cells to this toxin in the presence of fetal calf serum (FCS) decreased [3H]thymidine incorporation by 40% and induced a 40% increase in doubling time. This resulted in a 30% decrease in cell number per well after 48 h. These effects of B. pertussis toxin did not appear to be related to an increase in cyclic adenosine monophosphate (cAMP) concentration, because forskolin had the opposite effect on cell proliferation. Finally, B. pertussis toxin prevented serum-induced Raf1 association to the plasma membrane, possibly by disrupting FCS-induced G beta gamma effects on the Ras/Raf1 pathway. Since Go alpha and Gi alpha 1 subunits were absent in preadipocytes, we conclude that Gi2 and/or Gi3 proteins transduce some mitogenic signals of FCS through release of G beta gamma subunits. The subcellular distribution of Gi alpha 2 and Gi alpha 3 suggests that part of their functions result from interactions with components other than the plasma membrane.

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.

In vivo desensitization of the beta, but not the alpha 2-adrenoreceptor-coupled-adenylate cyclase system in hamster white adipocytes after administration of epinephrine.

To assess the physiological relevance of the changes in adrenergic receptor observed in adipocyte membranes after in vitro exposure to catecholamines, hamster white adipocytes, which possess both beta- and alpha-adrenergic receptors, were studied after a 6-day in vivo epinephrine administration (1 mg/kg, daily). This treatment resulted in a 3-fold increase in total plasma catecholamine levels and in the following changes in the adipocytes. The lipolytic and cAMP responses to beta-agonists, ACTH, and theophylline were decreased by 55-60%, but the sensitivity of these responses to (-)isoproterenol was unchanged. Although basal adenylate cyclase activity was unaffected, (-)isoproterenol-, ACTH- or fluoride-stimulated activities were reduced by half, a defect which persisted in the presence of guanosine 5'-[beta, gamma-imido]triphosphate. Furthermore, the number of beta-adrenergic receptors was also decreased by 54%. In contrast, epinephrine treatment failed to impair the adipocyte antilipolytic response to the alpha 2-agonist clonidine, the alpha 2-component of epinephrine, and the adenosine analog N6-phenylisopropyladenosine, the adenylate cyclase inhibitory response to these compounds, and the number of alpha 2-adrenergic receptors. These results indicate that in vivo epinephrine administration does not alter the alpha 2-adrenergic system of hamster white adipocyte, but desensitizes the lipolytic response to beta-agonists and also to nonadrenergic lipolytic agents. It is therefore suggested that the likely mechanism(s) responsible for this lipolysis desensitization mainly consists in impaired adenylate cyclase coupling and possibly in altered intracellular processes, rather than in the observed beta-adrenergic receptor down-regulation.

Estradiol treatment decreases the lipolytic responses of hamster white adipocytes through a reduction in the activity of the adenylate cyclase catalytic subunit.

After 5 days of daily administration of 10 micrograms estradiol to 6-week-old male hamsters, the in vitro maximal lipolytic and cAMP responses of white adipocytes to isoproterenol, epinephrine, ACTH, and theophylline were reduced by one half, with no change in the sensitivity of these responses. In contrast, the antilipolytic response to the alpha 2-adrenergic agonist clonidine was unimpaired. beta-Adrenergic receptor number and affinity, assessed in intact cells with [3H]CGP-12177 binding, showed no difference between control and estradiol-treated hamsters. In adipocyte membranes from estradiol-treated hamsters, maximal adenylate cyclase responses to Mn2+, GTP alone or in combination with isoproterenol, ACTH, or fluoride were all decreased by 30-40% below the values found in controls, but the sensitivity of these responses was unaltered. The maximum velocity (Vmax) of adenylate cyclase was reduced by one half in estrogen-treated animals, but the Michaelis-Menten constant (Km) of the enzyme for ATP was unchanged. Finally, complementation of adipocyte membranes with solubilized human erythrocyte Ns failed to restore to control values the maximal adenylate cyclase response to isoproterenol plus guanosine 5'-[beta, gamma-imido]-triphosphate in the estradiol-treated hamsters. These results indicate that a defect in the catalytic subunit of adenylate cyclase is one of the mechanisms through which estradiol treatment reduces the lipolytic response of hamster white adipocytes.

Influence of ovariectomy and regional fat distribution on the membranous transducing system controlling lipolysis in rat fat cells.

The mechanism by which sex steroid hormones modulate the lipolytic process in fat cells is still not completely understood, particularly at the level of the membrane. As estrogens seem to have regional influence on adipose tissue, we have compared the effects of long term ovariectomy (3 weeks) on the membranous transducing system controlling lipolysis in two rat fat deposits, the femoral sc and the parametrial adipose tissue. These experiments demonstrate the following. 1) Compared to parametrial adipocytes, sc adipocytes have a reduced cAMP production and a decreased beta-adrenoceptor number associated with a reduced lipolytic activity. 2) In parametrial adipocytes, ovariectomy induces a decrease in cAMP production and in both the beta-agonist-stimulated response and the catalytic activity of adenylate cyclase in association with a decreased lipolysis. In contrast, these parameters are unaltered by ovariectomy in sc adipocytes. This study, which reveals site-related differences in the sensitivity of the fat cell adenylate cyclase system to ovarian status, suggests that these differences may explain some of the sex-related regional specificities of adipose tissue metabolism and distribution.

Control of rat preadipocyte adipose conversion by ovarian status: regional specificity and possible involvement of the mitogen-activated protein kinase-dependent and c-fos signaling pathways.

As ovariectomy induces obesity in rats, we have investigated the influence of ovariectomy and hormone replacement on the proliferation and differentiation capacities of rat cultured preadipocytes removed from different fat depots (femoral sc, parametrial, and perirenal). Ovariectomy induced increased proliferation and differentiation as well as high mitogen-activated protein (MAP) kinase activity and c-fos protein induction in both confluent and differentiated preadipocytes from perirenal fat depots. In parametrial preadipocytes, ovariectomy also increased proliferation and c-fos protein induction, but failed to alter the capacities of these cells to differentiate. Treatment of ovariectomized rats with estradiol and progesterone reversed the promoting effect of ovariectomy on proliferation, differentiation, and c-fos induction in perirenal preadipocytes, but not the MAP kinase activation observed during the proliferative phase. This treatment also reversed the promoting effect of ovariectomy on proliferation and c-fos induction seen in confluent parametrial preadipocytes. In contrast, sc preadipocytes were totally insensitive to ovarian status in terms of proliferation and differentiation capacities, MAP kinase activity, and c-fos induction. This study demonstrates that adipogenesis is site-specifically controlled by the ovarian status in the rat. It also suggests that ovariectomy-induced obesity (mainly abdominal) could be related to changes in some of the signaling pathways controlling adipogenesis in intraabdominal preadipocytes.

Influence of androgenic status on the alpha 2/beta-adrenergic control of lipolysis in white fat cells: predominant alpha 2-antilipolytic response in testosterone-treated-castrated hamsters.

The aim of this study was to evaluate the influence of castration with or without testosterone propionate (TP) administration (one daily injection of 1 mg for 10 days) on the fat cell lipolytic activity in male hamsters. Basal and maximal lipolytic responses to the pure beta-adrenergic agonist isoproterenol, the mixed alpha 2-and beta-adrenergic agonist epinephrine, and the nonadrenergic compounds ACTH and 3-isobutyl-1-methylxanthine were all reduced by half in castrated animals. TP treatment restored these defective responses to control values, except the response to epinephrine which remained paradoxically unchanged. Sensitivity of lipolysis to epinephrine was unimpaired by castration but markedly reduced (10-fold) in TP-treated castrated hamsters. The antilipolytic potencies of the alpha 2-component of epinephrine and of the two alpha 2-agonists, UK 14304 and clonidine, were reduced by half in castrated animals, and returned to a value slightly higher than control after TP treatment. These changes in lipolysis were accompanied by parallel alterations in the stimulated cAMP responses to isoproterenol and forskolin but not to epinephrine. The latter was either unimpaired by castration or was clearly inhibited after TP treatment. Castration also induced a 2-fold decrease in the inhibitory potency of clonidine toward forskolin-stimulated cAMP production. Finally, these changes in the potency of clonidine were accompanied by parallel variations of the number of fat cell alpha 2-adrenoreceptors. These results indicate that testosterone in vivo, while increasing the beta-adrenergic lipolytic action of catecholamines (possibly through enhancement of the adenylate cyclase activity), promotes, to a greater extent, their alpha 2-adrenoreceptor-mediated antilipolytic potency. By providing the first demonstration that the androgenic status controls the functional alpha 2/beta-adrenergic balance in fat cells, this study also emphasizes the potential importance of such a control in the mechanisms underlying the sex-related differences in adipose tissue regional distribution and fat cell size.