isolation-differentiation-primary-white-brown-preadipocytes-from

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

The Scripps Research Institute, La Jolla, CA, USA

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.

TLE3 is a dual-function transcriptional coregulator of adipogenesis.

Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. 

Application of activity-based protein profiling to study enzyme function in adipocytes.

Diterpene derivatives of the natural product acanthoic acid have potent anti-inflammatory effects in vivo. In this issue of Chemistry & Biolgy, Través and colleagues report that the primary molecular mechanism of action of diterpenes structurally related to acanthoic acid is the direct activation of PI3-kinase signaling in macrophages, which in turn inhibits NF-κB activation and suppresses proinflammatory gene expression. 

Activating PI3-kinase to dampen inflammation.

Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis. 

ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue.

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

Ligand and Target Discovery by Fragment-Based Screening in Human Cells.

Extracts of the hops plant have been shown to reduce weight and insulin resistance in rodents and humans, but elucidation of the mechanisms responsible for these benefits has been hindered by the use of heterogeneous hops-derived mixtures. Because hop extracts are used as flavoring agents for their bitter properties, we hypothesized that bitter taste receptors (Tas2rs) could be mediating their beneficial effects in metabolic disease. Studies have shown that exposure of cultured enteroendocrine cells to bitter tastants can stimulate release of hormones, including glucagon-like peptide 1 (GLP-1). These findings have led to the suggestion that activation of Tas2rs may be of benefit in diabetes, but this tenet has not been tested. Here, we have assessed the ability of a pure derivative of a hops isohumulone with anti-diabetic properties, KDT501, to signal through Tas2rs. We have further used this compound as a tool to systematically assess the impact of bitter taste receptor activation in obesity-diabetes.

Intestinal bitter taste receptor activation alters hormone secretion and imparts metabolic benefits.

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

Enrique Saez

Department of Molecular Medicine

The Scripps Research Institute, La Jolla, CA, USA

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Enrique Saez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Medicine

The Scripps Research Institute, La Jolla, CA, USA

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Department of Molecular Medicine