SUMMARYObesity is characterized by an expansion of white adipose tissue mass that results from an increase in the size and the number of adipocytes. However, the mechanisms responsible for the formation of adipocytes during development and the molecular mechanisms regulating their increase and maintenance in adulthood are poorly understood. Here, we report the use of leptinluciferase BAC transgenic mice to track white adipose tissue (WAT) development and guide the isolation and molecular characterization of adipocytes during development using DNA microarrays. These data reveal distinct transcriptional programs that are regulated during murine WAT development in vivo. By using a de novo cis-regulatory motif discovery tool (FIRE), we identify two early gene clusters whose promoters show significant enrichment for NRF2/ETS transcription factor binding sites. We further demonstrate that Ets transcription factors, but not Nrf2, are regulated during early adipogenesis and that Ets2 is essential for the normal progression of the adipocyte differentiation program in vitro. These data identify ETS2 as a functionally important transcription factor in adipogenesis and its possible role in regulating adipose tissue mass in adults can now be tested. Our approach also provides the basis for elucidating the function of other gene networks during WAT development in vivo. Finally these data confirm that although gene expression during adipogenesis in vitro recapitulates many of the patterns of gene expression in vivo, there are additional developmental transitions in pre and post-natal adipose tissue that are not evident in cell culture systems.
The biological roles of cyclic nucleotide phosphodiesterase 11 (PDE11) enzymes are poorly understood, in part due to the lack of selective inhibitors. To address the need for such compounds, we completed an ~200,000 compound high-throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay, and identified 4 potent and selective PDE11 inhibitors. One compound, along with two structural analogs, elevates cAMP and cortisol levels in human adrenocortical cells, consistent with gene association studies that link PDE11 activity to adrenal function. As such, these compounds can immediately serve as chemical tools to study PDE11 function in cell culture, and as leads to develop therapeutics for the treatment of adrenal insufficiencies. Our results further validate this yeast-based HTS platform for the discovery of potent, selective, and biologically active PDE inhibitors.
Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor α from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.
Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High Throughput Screens (HTSs) for PDE inhibitors that possess ‘drug-like’ characteristics, displaying activity in a growth stimulation assay over a 48-hr period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme’s potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium in order to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify if different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.