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Background CD36 is a membrane glycoprotein, contributing to the pathogenesis of metabolic disorders, like obesity, which has become a major health concern worldwide. Methods A potential functional role of the scavenger receptor CD36 was investigated in in vitro adipocyte differentiation and in vivo adipogenesis. Results During differentiation of 3T3-F442A preadipocytes into mature adipocytes, expression of CD36 was upregulated and CD36 gene silencing resulted in impaired differentiation, as monitored by Oil Red O staining and expression of adipogenic markers. De novo fat pad formation in NUDE mice following injection of preadipocytes was significantly reduced upon CD36 gene silencing as compared to control. This was associated with marked adipocyte hypotrophy and reduced adipose tissue adipocyte content. Macrophage infiltration in de novo fat tissues derived from preadipocytes with CD36 gene silencing was not significantly different from controls. Collagen content was significantly higher in de novo fat with CD36 gene silencing. In a nutritionally induced obesity model, total body weight as well as subcutaneous and gonadal adipose tissue mass were significantly lower in CD36 deficient mice as compared to wild-type littermates. General significance Thus, our data support a functional role of CD36 in promoting adipogenesis in vitro as well as in vivo.

Type 2 diabetes is causally related to obesity and characterized by dysfunctional pancreatic beta cells. It is so far unclear whether direct interactions exist between adipocytes and beta cells and possibly raise any pathogenic relevance. In this study, we examined whether 9-day co-cultured 3T3-F442A (pre)adipocytes and primary rat pancreatic beta cells exert an influence on each other's function. In the presence of beta cells, 3T3-F442A cells became lipid-storing cells expressing markers of differentiated adipocytes and releasing adiponectin. This effect was attributed to the medium insulin levels (around 0.1 μM) and was associated with an elevated glucose consumption by the 3T3-F442A cells. The subsequent decrease in medium glucose concentration reduced the rate of insulin release by beta cells cultured at 10 mM glucose, and thus suppressed their degranulation during culture. These changes in beta cell function did not occur at 20 mM glucose and were reversible upon removal of the 3T3-F422A cells. They could not be reproduced by 3T3-F422A-conditioned medium containing varying adiponectin concentrations. These data indicate that insulin secreted by beta cells is sufficient to induce differentiation of preadipocytes without addition of exogenous adipogenic factors. Over 9 days culture, (pre)adipocytes did not directly and irreversibly affect beta cell functions.

The potential role of placental growth factor (PlGF) in early stages of adipogenesis was investigated in vivo using a murine model of obesity, as well as in vitro using cultured preadipocytes. PlGF-deficient (PlGF-/-) and wild-type (WT) mice, kept on high fat diet (HFD) for 3 weeks, had comparable body weight and weight of subcutaneous (SC) and gonadal (GON) adipose tissues. Blood vessel size and blood vessel density, normalized to adipocyte number, were not significantly different in SC and GON adipose tissues of both genotypes. Differentiation of embryonic fibroblasts derived from WT or PlGF-/- mice into mature adipocytes was comparable. Furthermore, addition of recombinant PlGF, of the PlGF neutralizing MAb PL5D11D4 or of the anti-Flk-1 MAb DC101 to cultured 3T3-F442A preadipocytes did not significantly affect their differentiation into mature adipocytes. Ex vivo blood vessel outgrowth following seeding of adipose tissue-derived microvessel fragments in 3D-collagen gels was not affected by PlGF deficiency. Thus, in murine model systems, PlGF does not seem to play an important role in early adipogenesis.

Obesity is a common disorder and related diseases such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer are a major cause of mortality and morbidity in Western-type societies. Development of obesity is associated with extensive modifications in adipose tissue involving adipogenesis, angiogenesis and extracellular matrix proteolysis. The fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems cooperate in these processes. A nutritionally induced obesity model in transgenic mice has been used extensively to study the role of the fibrinolytic and MMP systems in the development of obesity. These studies support a role of both systems in adipogenesis and obesity; the role of specific members of these families, however, remains to be determined.

A low-molecular-weight receptor tyrosine kinase inhibitor, 1-(6,7-dihydro-5H-benzo(6,7)cyclohepta(1,2-c)pyridazin-3-yl)-N3-((7-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo(7)annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine (R428) with high affinity and selectivity for the growth arrest-specific protein 6 (GAS6) receptor Axl was used to study a potential role of GAS6 signaling in adiposity. In vitro, R428 caused a concentration-dependent inhibition of preadipocyte differentiation into mature adipocytes, as evidenced by reduced lipid uptake. Inhibition of Axl-mediated signaling was confirmed by reduced levels of phospho-Akt activity. In vivo, oral administration of R428 for 5 weeks to mice kept on a high-fat diet resulted in significantly reduced weight gain and subcutaneous and gonadal fat mass. This was associated with marked adipocyte hypotrophy, enhanced macrophage infiltration, and apoptosis. Thus, affecting GAS6 signaling through receptor antagonism using a low-molecular-weight Axl antagonist impairs adipocyte differentiation and reduces adipose tissue development in a murine model of nutritionally induced obesity.

Summary. Objective: To substantiate a potential role of plasminogen activator inhibitor-1 (PAI-1) in adipogenesis, we have studied its effects on in vitro adipocyte differentiation and on in vivo adipose tissue formation. Results: Our in vitro data do not support a functional role of PAI-1, as substantiated by our findings that: (i) inhibition of PAI-1 with a neutralizing antibody did not affect differentiation of 3T3-F442A preadipocytes; (ii) overexpression of murine PAI-1 in 3T3-F442A cells had no effect on differentiation; and (iii) differentiation of PAI-1-deficient murine embryonic fibroblasts into mature adipocytes was comparable to wild-type (WT) cells. Furthermore, our in vivo studies did not reveal an important role for PAI-1, as suggested by our findings that: (i) de novo fat pad formation in NUDE mice following injection of 3T3-F442A cells was not affected by a PAI-1 neutralizing antibody; and (ii) adipose tissue formation following combined injection of Matrigel and basic fibroblast growth factor was comparable in WT and in PAI-1 deficient mice. Conclusion: Taken together, these in vitro and in vivo studies in murine model systems do not support an important functional role of PAI-1 in adipogenesis.

Placental growth factor (PlGF)-deficient (PlGF-/-) and wild-type mice were kept on a standard-fat or high-fat diet for 15 weeks. With the standard-fat diet, the body weights of PlGF-/- and wild-type mice were comparable, whereas the combined weight of subcutaneous and gonadal adipose tissues was lower in PlGF-/- mice (P = 0.02). With the high-fat diet, PlGF-/- mice had a lower body weight (P < 0.05) and less total subcutaneous plus gonadal adipose tissue (P < 0.0001). Blood vessel size was lower in gonadal adipose tissue of PlGF-/- mice with both the standard-fat and high-fat diet (P < 0.05). Blood vessel density, normalized to adipocyte number, was significantly lower in subcutaneous adipose tissue of PlGF-/- mice fed the high-fat diet (P < 0.01). De novo adipose tissue development in nude mice injected with 3T3-F442A preadipocytes was reduced (P < 0.005) by administration of a PlGF-neutralizing antibody. Bone marrow transplantation from wild-type or PlGF-/- mice to wild-type or PlGF-/- recipient mice revealed significantly lower blood vessel density in PlGF-/- recipient mice without an effect on adipose tissue growth. Thus, in murine models of diet-induced obesity, inactivation of PlGF impairs adipose tissue development, at least in part as a result of reduced angiogenesis. ispartof: Diabetes vol:55 issue:10 pages:2698-2704 status: published

Endocrine disruptors pose a growing threat to human and wildlife health. Validated test systems are required to study the mechanisms by which chemicals may interfere with the endocrine system. In order to identify compounds with (anti)androgenic activity, we used several in vitro bioassays, based on different androgen receptor (AR) functions, including AR transactivation and interaction between the amino-terminal domain and the ligand-binding domain. The AR activity assay, based on activation of the transcription of an androgen-responsive reporter gene in the presence of androgen, proved to excel in terms of high fold induction range and low minimal detection limit. The EC50 value, defined as the concentration that leads to half the maximal response and reflecting the potency of the synthetic androgen R1881 (methyltrienolone), was found to be 250 pM, consistent with the high affinity of this ligand to the human AR. A number of environmental samples were tested in the bioassay. The bioassay also could be used to detect antiandrogenic activity, because known AR-antagonists were able to inhibit R1881-mediated transactivation.

The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.