Summary Nutritional and pharmacological stimuli can dramatically alter the cellular phenotypes in white adipose tissue (WAT). Utilizing genetic lineage tracing techniques, we demonstrate that brown adipocytes (BA) that are induced by β3-adrenergic receptor activation in abdominal WAT arise from the proliferation and differentiation of cells expressing platelet-derived growth factor receptor alpha (PDGFRα), CD34 and Sca1 (PDGFRα+ cells). PDGFRα+ cells have a unique morphology in which extended processes contact multiple cells in the tissue microenvironment. Surprisingly, these cells also give rise to white adipocytes (WA) that can comprise up to 25% of total fat cells in abdominal fat pads following 8 weeks of high fat feeding. Isolated PDGFRα+ cells differentiated into both BA and white adipocytes (WA) in vitro, and generated WA after transplantation in vivo. The identification of PDGFRα+ cells as bipotential adipocyte progenitors will enable further investigation of mechanisms that promote therapeutic cellular remodeling in adult WAT.

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity and inflammatory response. PER2-deficient mice display altered lipid metabolism, with drastic reduction of total triacylglycerol and non-esterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated to altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the pro-adipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention.

The mobilization of stored lipid by hormones is a fundamental function of fat cells, and there is strong evidence that perilipin (Plin), a lipid droplet scaffold, and adipose tissue triglyceride lipase (Atgl), a triglyceride-specific lipase, play critical roles. Previous work suggested that Abhd5, a protein activator of Atgl, coordinates with Plin in controlling basal and stimulated lipolysis; however, the underlying mechanism is controversial. Growing evidence indicates that hormone-stimulated lipolysis involves protein trafficking and specific protein-protein interactions at the surface of lipid droplets (LDs), 2 and there is strong evidence that perilipin A (Plin), a lipid droplet scaffold protein, plays a central role in orchestrating interactions among lipolytic effector proteins (1, 2). For example, phosphorylated Plin provides a docking site by which phosphorylated hormone-sensitive lipase (HSL) gains access to substrates at the surface of LDs (3-5). Adipose triglyceride lipase (Atgl, also known as desnutrin, Pnpla2) also participates in protein kinase A (PKA)-stimulated lipolysis (6, 7), and Plin is thought to be involved in this regulation (8); however, the mechanisms involved are not understood, and proposed models that address this regulation disagree.Unlike HSL, Atgl is not a direct target of PKA phosphorylation, and therefore, the mechanism of its activation must be indirect. One model whereby Plin could indirectly regulate Atgl activity is by controlling availability of its co-activator, Abhd5 (also known as CGI-58), in a manner that depends on PKA-dependent phosphorylation of Plin (3). According to this "sequestration-release" model, Plin sequesters Abhd5 in the basal state, thereby preventing activation of Atgl and suppressing basal lipolysis. PKA activation leads to Plin phosphorylation, release of Abhd5, and subsequent activation of Atgl. This mechanism is supported by biochemical and dynamic imaging experiments demonstrating that Abhd5 binds Plin in the unstimulated state, and PKA-mediated phosphorylation of Plin leads to rapid release of Abhd5 from Plin (3, 9 -11). Additionally, knockdown of Abhd5 reduces both basal and stimulated lipolysis (11, 12), although whether this effect involves interactions with Plin has been questioned (12).Although these data are consistent with the general model, there are no data demonstrating that 1) the interaction of Abhd5 with Plin and Atgl is mutually exclusive (that is, Plin suppresses the interaction of Abhd5 with Atgl) or that 2) phosphorylation of Plin promotes the physical interaction of Abhd5 with Atgl. In fact, an alternative model has been described whereby Atgl regulates basal but not stimulated lipolysis (1). According to this model, basal lipolysis is stimulated by a ternary complex containing Plin, Abhd5, and Atgl. Furthermore, the model proposes that Abhd5 is not involved in PKA-stimulated activation of Atgl because it is released into the cytoplasm upon Plin phosphorylation.In the experiments detailed below, we examined the functional interact...

Recruitment of brown/beige adipocytes (BAs) in white adipose tissue (WAT) involves proliferation and differentiation of adipocyte stem cells (ASCs) in concert with close interactions with resident immune cells. To deconvolve stromal cell heterogeneity in a comprehensive and unbiased fashion, we performed single-cell RNA sequencing (scRNA-seq) of >33,000 stromal/vascular cells from epididymal WAT (eWAT) and inguinal WAT (iWAT) under control conditions and during β3-adrenergic receptor (ADRB3) activation. scRNA-seq identified distinct ASC subpopulations in eWAT and iWAT that appeared to be differentially poised to enter the adipogenic pathway. ADRB3 activation triggered the dramatic appearance of proliferating ASCs in eWAT, whose differentiation into BAs could be inferred from a single time point. scRNA-seq identified various immune cell types in eWAT, including a proliferating macrophage subpopulation that occupies adipogenic niches. These results demonstrate the power of scRNA-seq to deconstruct adipogenic niches and suggest novel functional interactions among resident stromal cell subpopulations.

SUMMARY The regulatory events guiding progenitor activation and differentiation in adult white adipose tissue are largely unknown. We report that induction of brown adipogenesis by β3-adrenergic receptor (ADRB3) activation involves the death of white adipocytes and their removal by M2-polarized macrophages. Recruited macrophages express high levels of osteopontin (OPN), which attracts a subpopulation of PDGFRα+ progenitors expressing CD44, a receptor for OPN. Preadipocyte proliferation is highly targeted to sites of adipocyte clearance and occurs almost exclusively in the PDGFRα+ CD44+ subpopulation. Knockout of OPN prevents formation of crown-like structures by ADRB3 activation and the recruitment, proliferation, and differentiation of preadipocytes. The recruitment and differentiation of PDGFRα+ progenitors are also observed following physical injury, during matrix-induced neogenesis, and in response to high-fat feeding. Each of these conditions recruits macrophages having a unique polarization signature, which may explain the timing of progenitor activation and the fate of these cells in vivo.

A central function of adipocytes is the storage and mobilization of energy in the form of triglyceride. A considerable amount is known about the enzymatic basis for lipogenesis and lipolysis; however, our understanding of how these processes are organized and regulated within cells is incomplete. Until recently, cellular triglyceride was considered to be stored in droplets lacking biological structure or organization. Growing evidence, however, suggests that lipid droplets are specialized, heterogeneous organelles that perform distinct roles in lipid biosynthesis, transport, and mobilization (1-3). A large number of proteins have been found to change their associations with lipid droplets in response to lipolytic stimulation. How these proteins are coordinated to control lipid storage and utilization remains largely unknown.It is well established that activation of cAMP-dependent kinase (PKA) 3 is the major signaling mechanism by which hormones and neurotransmitters stimulate lipolysis in adipocytes (4, 5). Most work regarding hormone stimulated lipolysis has focused on perilipin (Plin), a protein that binds to the surface of certain lipid droplets (LDs), and hormone-sensitive lipase (HSL), which translocates to lipid during lipolytic activation.Plin is the major target for PKA-mediated phosphorylation in adipocytes (6, 7), appears to be essential for hormone-stimulated lipolysis (8) and exerts both positive and negative effects on lipolytic rate (9 -11). Plin was originally viewed as a physical barrier that passively regulated access of lipases to store triglyceride; however, more recent data indicates that Plin may function more as a LD scaffold that directs the trafficking of lipolytic proteins to a specialized subcellular domain (11). Nonetheless, the temporal and spatial relationships between Plin and other proteins in the lipolytic pathway during PKA activation remain ill-defined.PKA activation promotes the translocation of HSL to lipid, which correlates with the magnitude of stimulated lipolysis (7,12). Work from several laboratories indicates that Plin and HSL synergize to trigger PKA-mediated lipolysis (11, 13), and our recent work indicates that HSL specifically translocates to a subset of LDs containing Plin (11). It is presently unclear whether Plin and HSL interact directly, whether the interaction takes place on certain LDs and not others, or whether the interaction occurs on a timescale that is compatible with the initiation of lipolysis.The complexity of regulated lipolysis has recently expanded with the identification of adipose triglyceride lipase (Atgl) (14, * This work was supported in part by grants from the National Institutes of Health (DK 62292), the American Diabetes Association, the Fund for Medical Research and Education at WSU, and the Joseph Young Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fac...

This work investigated how cold stress induces the appearance of brown adipocytes (BAs) in brown and white adipose tissues (WATs) of adult mice. In interscapular brown adipose tissue (iBAT), cold exposure increased proliferation of endothelial cells and interstitial cells expressing platelet-derived growth factor receptor, α polypeptide (PDGFRα) by 3- to 4-fold. Surprisingly, brown adipogenesis and angiogenesis were largely restricted to the dorsal edge of iBAT. Although cold stress did not increase proliferation in inguinal white adipose tissue (ingWAT), the percentage of BAs, defined as multilocular adipocytes that express uncoupling protein 1, rose from undetectable to 30% of total adipocytes. To trace the origins of cold-induced BAs, we genetically tagged PDGFRα(+) cells and adipocytes prior to cold exposure, using Pdgfra-Cre recombinase estrogen receptor T2 fusion protein (CreER(T2)) and adiponectin-CreER(T2), respectively. In iBAT, cold stress triggered the proliferation and differentiation of PDGFRα(+) cells into BAs. In contrast, all newly observed BAs in ingWAT (5207 out of 5207) were derived from unilocular adipocytes tagged by adiponectin-CreER(T2)-mediated recombination. Surgical denervation of iBAT reduced cold-induced brown adipogenesis by >85%, whereas infusion of norepinephrine (NE) mimicked the effects of cold in warm-adapted mice. NE-induced de novo brown adipogenesis in iBAT was eliminated in mice lacking β1-adrenergic receptors. These observations identify a novel tissue niche for brown adipogenesis in iBAT and further define depot-specific mechanisms of BA recruitment.

Selective agonists of beta(3)-adrenergic receptors (Adrb3) exhibit potent anti-diabetes properties in rodent models when given chronically, yet the mechanisms involved are poorly understood. A salient feature of chronic Adrb3 activation is pronounced remodeling of white adipose tissue (WAT), which includes mitochondrial biogenesis and elevation of metabolic rate. To gain insights into potential mechanisms underlying WAT remodeling, the time course of remodeling induced by the Adrb3 agonist CL-316,243 (CL) was analyzed using histological, physiological, and global gene profiling approaches. The results indicate that continuous CL treatment induced a transient proinflammatory response that was followed by cellular proliferation among stromal cells and multilocular adipocytes. CL treatment strongly fragmented the central lipid storage droplet of mature adipocytes and induced mitochondrial biogenesis within these cells. Mitochondrial biogenesis was correlated with the upregulation of genes involved in fatty acid oxidation and mitochondrial electron transport activity. The elevated catabolic activity of WAT was temporally correlated with upregulation of peroxisome proliferator-activated receptor-alpha and its target genes, suggesting involvement of this transcription factor in coordinating the gene program that elevates WAT catabolic activity.