Human adipocyte function is impacted by mechanical cues.

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity-induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri-adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL-6, G-CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro-inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri-adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT-dependent CTGF promoter activity, which depends on ß1-integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co-activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT-induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation.

Semaphorin 3C is a novel adipokine linked to extracellular matrix composition.

AIMS/HYPOTHESIS: Alterations in white adipose tissue (WAT) function, including changes in protein (adipokine) secretion and extracellular matrix (ECM) composition, promote an insulin-resistant state. We set out to identify novel adipokines regulated by body fat mass in human subcutaneous WAT with potential roles in adipose function. METHODS: Adipose transcriptome data and secretome profiles from conditions with increased/decreased WAT mass were combined. WAT donors were predominantly women. In vitro effects were assessed using recombinant protein. Results were confirmed by quantitative PCR/ELISA, metabolic assays and immunochemistry in human WAT and adipocytes. RESULTS: We identified a hitherto uncharacterised adipokine, semaphorin 3C (SEMA3C), the expression of which correlated significantly with body weight, insulin resistance (HOMA of insulin resistance [HOMAIR], and the rate constant for the insulin tolerance test [KITT]) and adipose tissue morphology (hypertrophy vs hyperplasia). SEMA3C was primarily found in mature adipocytes and had no direct effect on human adipocyte differentiation, lipolysis, glucose transport or the expression of ß-oxidation genes. This could in part be explained by the significant downregulation of its cognate receptors during adipogenesis. In contrast, in pre-adipocytes, SEMA3C increased the production/secretion of several ECM components (fibronectin, elastin and collagen I) and matricellular factors (connective tissue growth factor, IL6 and transforming growth factor-ß1). Furthermore, the expression of SEMA3C in human WAT correlated positively with the degree of fibrosis in WAT. CONCLUSIONS/INTERPRETATION: SEMA3C is a novel adipokine regulated by weight changes. The correlation with WAT hypertrophy and fibrosis in vivo, as well as its effects on ECM production in human pre-adipocytes in vitro, together suggest that SEMA3C constitutes an adipocyte-derived paracrine signal that influences ECM composition and may play a pathophysiological role in human WAT.

Using gene expression to predict the secretome of differentiating human preadipocytes.

OBJECTIVE: To characterize the secretome of differentiating human preadipocytes using global gene expression profiling. DESIGN: Gene expression was measured using microarrays at days 0, 1, 3, 5, 7 and 10 in primary preadipocytes undergoing adipogenesis (n=6 independent subjects). Predictive bioinformatic algorithms were employed to identify those differentially expressed genes that code for secreted proteins. MEASUREMENTS: Gene expression was assessed using microarrays and real-time reverse transcriptase PCR, bioinformatic predictive algorithms were used to identify the secretome of differentiating preadipocytes, and the secretion of the most significant candidates were confirmed at the protein level using western blots or ELISA tests. Gene expression was also assayed in the adipocyte and stroma vascular fraction (SVF) of obese subjects. RESULTS: Microarray analysis identified 33 genes whose expression significantly changed (false discovery rate of 1%) during adipogenesis and code for secreted proteins. Of these genes, 18 are novel candidate adipose tissue 'secretome' genes. Their relative gene expression in adipocyte and SVF of obese subjects revealed that most of these genes are more highly expressed in the SVF. A novel candidate, matrix gla protein (MGP), was upregulated (approximately 30-fold) during adipogenesis, second only to leptin (approximately 50-fold). MGP and another secretome candidate protein, inhibin beta B (INHBB), were detected in the secretion media of adipocytes isolated from adipose tissue explants. CONCLUSIONS: Gene expression coupled with predictive bioinformatic algorithms has proved a valid and alternative approach to further define the adipocyte secretome. Many of the novel candidate secretome genes are components of the coagulation and fibrinolytic systems. MGP and INHBB represent new adipokines whose function in adipose tissue remains to be unravelled.

Interaction of PTPB with the insulin receptor precursor during its biosynthesis in the endoplasmic reticulum.

PTP1B is a protein tyrosine-phosphatase predominantly located on the cystosolic surface of the endoplasmic reticulum. This tyrosine-phosphatase plays a major role in the regulation of the activity of the insulin receptor (IR). We have studied the interaction of the IR with PTP1B in living cells using bioluminescence resonance energy transfer (BRET). The IR was fused to Renilla luciferase and a substrate-trapping mutant of PTP1B was fused to the yellow variant of the green fluorescent protein (YFP). When the two partners interacted, an energy transfer occurred between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, could be detected. The interaction of the IR with PTP1B could be monitored in real time for more than 30 min. Insulin rapidly and dose-dependently stimulated this interaction. The basal (insulin-independent) interaction of IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B, indicating that this basal interaction mainly occurred in the endoplasmic reticulum. In the basal state, PTP1B and the IR indeed co-localized in the endoplasmic reticulum, as demonstrated by confocal microscopy and cell fractionation experiments. Moreover, inhibition of IR processing with tunicamycin indicated that the basal interaction of PTP1B with IR occurred during biosynthesis of the IR precursor in the endoplasmic reticulum. These results strongly suggest that PTP1B not only dephosphorylates the insulin receptor that has been activated by insulin, but also regulates the insulin receptor precursor during its biosynthesis. Localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the immature insulin receptor precursor.

Looking for an insulin pill? Use the BRET methodology!

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.

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.

Progesterone stimulates adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c gene expression. potential mechanism for the lipogenic effect of progesterone in adipose tissue.

Fatty acid synthase (FAS), a nutritionally regulated lipogenic enzyme, is transcriptionally controlled by ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), through insulin-mediated stimulation of ADD1/SREBP1c expression. Progesterone exerts lipogenic effects on adipocytes, and FAS is highly induced in breast tumor cell lines upon progesterone treatment. We show here that progesterone up-regulates ADD1/SREBP1c expression in the MCF7 breast cancer cell line and the primary cultured preadipocyte from rat parametrial adipose tissue. In MCF7, progesterone induced ADD1/SREBP1c and Metallothionein II (a well known progesterone-regulated gene) mRNAs, with comparable potency. In preadipocytes, progesterone increased ADD1/SREBP1c mRNA dose-dependently, but not SREBP1a or SREBP2. Run-on experiments demonstrated that progesterone action on ADD1/SREBP1c was primarily at the transcriptional level. The membrane-bound and mature nuclear forms of ADD1/SREBP1 protein accumulated in preadipocytes cultured with progesterone, and FAS induction could be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1 in these cells. Finally, in the presence of insulin, progesterone was unable to up-regulate ADD1/SREBP1c mRNA in preadipocytes, whereas its effect was restored after 24 h of insulin deprivation. Together these results demonstrate that ADD1/SREBP1c is controlled by progesterone, which, like insulin, acts by increasing ADD1/SREBP1c gene transcription. This provides a potential mechanism for the lipogenic actions of progesterone on adipose tissue.

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.

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.

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.

Modulation of white adipose tissue lipolysis by nitric oxide.

In isolated adipocytes, the nitrosothiols S-nitroso-N-acetyl-penicillamine (SNAP) and S-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 Km, 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 beta-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.

Site-related specificities of the control by androgenic status of adipogenesis and mitogen-activated protein kinase cascade/c-fos signaling pathways in rat preadipocytes.

In rats, castration induces a complete defective adipose conversion of preadipocytes from the epididymal fat depots (Lacasa, D., B. Agli, D. Noynarol, and Y. Giudicelli, 1995, Endocrine 3: 789-793). The aim of this study was to establish the eventual site-specificity of this effect as well as the mechanisms involved. Therefore, the influence of androgenic status on the Fos protein induction and the Raf/mitogen-activated protein (MAP) kinase kinase (MEK)MAP cascade, which are all required for adipose conversion of preadipocytes, was compared in proliferating and differentiated preadipocytes from femoral sc and deep intraabdominal (epididymal and perirenal) fat depots. In epididymal and perirenal proliferating preadipocytes, increased proliferation due to castration is associated with increased MAP kinase activity. However, higher immunoreactive levels of the upstream activators of MAP kinase, Raf-1 and MEK, were observed only in epididymal cells. Moreover, in vivo testosterone treatment corrected the effects of castration on Raf-1 but not on MEK and MAP kinase. MAP kinase activity was decreased during the course of adipogenesis. In differentiated cells, MAP kinase activity showed variations according to the anatomical origin of preadipocytes but not to the androgenic status. In contrast, MEK and Raf-1 immunoreactive levels were both sensitive to androgenic status but were differently affected depending on cell origin. Finally, the defective adipogenesis seen in epididymal preadipocytes from castrated rats was associated with reduced Fos protein induction in these cells, an alteration which was partly corrected by testosterone-treatment. Taken together, these results suggest that androgenic status affects adipogenesis from deep intraabdominal preadipocytes through alterations of some components of the MAP kinase cascade/Fos signaling pathways.

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.

Site-related differences in G-protein alpha subunit expression during adipogenesis in vitro: possible key role for Gq/11 alpha in the control of preadipocyte differentiation.

G protein alpha subunits were compared by immunoblotting in preadipocytes and adipocytes from rat subcutaneous and epididymal adipose tissue at three steps of adipogenesis. The most striking difference concerned the Gq/11 alpha subunits whose expression decreased during preadipocyte differentiation in subcutaneous cells while it remained constant in epididymal cells. The PKC inhibitor CGP 41251 increased glycerol 3-phosphate dehydrogenase activity (EC 1.1.1.8), a late marker of differentiation, in epididymal preadipocytes but not in subcutaneous cells. There was no difference in the proliferation capacities between subcutaneous and epididymal preadipocytes: in both cells, CGP 41251 led to the same decrease in [(3)H]thymidine incorporation and, at confluence, the amounts of Gq/11 alpha subunits were equivalent. No major site-related difference was found in the amounts of Gs and Gi alpha during adipogenesis. Thus, compared to epididymal preadipocytes, the higher capacity of subcutaneous preadipocyte to differentiate seems to be correlated with the decrease in Gq/11 alpha expression and the decreased Gq/11 mediated PKC activation.

Zeta PKC in rat preadipocytes: modulation by insulin and serum mitogenic factors and possible role in adipogenesis.

Several PKC isoforms belonging to the three PKCs' subfamilies cPKC alpha and beta isoforms, nPKC epsilon isoform and aPKC zeta isoform were detected by Western blot in rat preadipocytes. zeta PKC which appears involved in proliferation and differentiation of some cellular types was shown to display variations according to the preadipocyte anatomical origin and stage of differentiation. A rapid increase in zeta PKC in the cytosolic compartment and translocation into the nucleus were induced by mitogenic factors in proliferating preadipocytes and by insulin in differentiating preadipocytes. These findings suggest that zeta PKC could be involved i) in the post-receptor signaling pathway of serum mitogenic factors and insulin in preadipocytes, and ii) in the mechanisms underlying the variations in the proliferating and differentiating capacities of preadipocytes according to their anatomical localization.

Evidence for a regional-specific control of rat preadipocyte proliferation and differentiation by the androgenic status.

In the rat, castration induces a decreased weight of fat depots. One possible explanation for these alterations could be that the capacities of preadipocytes to proliferate and differentiate are reduced by castration. Considering the regional specification of adipose tissue metabolism, these capacities and their eventual modulation by the androgenic status were presently compared in cultured preadipocytes from rat subcutaneous (SC) and epididymal fat depots.In epididymal preadipocytes, castration induced an increase in their proliferative capacity and conversely, a decrease in their adipogenesis.In vivo treatment by testosterone reversed the proliferative alteration but not the defective adipogenesis caused by castration.In vitro, no direct effect of testosterone on the proliferative capacities of epididymal preadipocytes could be observed suggesting that testosterone acts indirectly or needs the presence of other cofactors, such as insulin, dexamethasone and growth hormone. Surprisingly, testosterone partly counteracted the inhibitory effect of growth hormone on preadipocyte differentiation.In contrast to these observations, SC preadipocytes were completely insensitive to the androgenic status in terms of proliferation and differentiation.This study showing site-specific effects of castration on preadipocyte proliferation and differentiation suggests that part of the decreased fatness induced by castration in the rat is related to the modulatory effect of androgenic status on adipogenesis in some deep fat depots.

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.

Influence of ovarian status and regional fat distribution on protein kinase C in rat fat cells.

The effects of ovarian status on insulin- and phorbol 12-myristate 13-acetate (TPA)-stimulated lipogenic responses, phorbol ester-specific binding to protein kinase C (PKC) and immunoblot-quantified beta- and epsilon-PKC isoform levels were compared in female rat fat cells from subcutaneous and deep intra-abdominal (parametrial) fat deposits. In control rats, the cytosolic PKC content per cell was 70% lower in subcutaneous than in parametrial adipocytes. In subcutaneous and parametrial fat cells, the cytosolic PKC contents were reduced by ovariectomy and restored to normal by the administration of ovarian hormones (oestradiol plus progesterone). However, the lipogenic response to TPA was unaltered by ovarian status in both fat deposits, contrasting with the insulin-stimulated lipogenic response. This response increased in parametrial fat cells after ovariectomy and returned to normal after ovarian hormone treatment whereas, in subcutaneous fat cells, the insulin response was either unaltered by ovariectomy or increased by the administration of ovarian hormones. This study shows important site-related differences in fat cell PKC content but also reveals a similar modulation of this PKC by ovarian status, regardless of the anatomical localization of the fat cells. The physiological significance of these findings is discussed.