Unraveling the complexity of thermogenic remodeling of white fat reveals potential antiobesity therapies.

Adipose tissue is a complex organ consisting of a mixture of mature adipocytes and stromal vascular cells. It displays a remarkable ability to adapt to environmental and dietary cues by changing its morphology and metabolic capacity. This plasticity is demonstrated by the emergence of interspersed thermogenic beige adipocytes within white depots in response to catecholamines secretion. Coordinated cellular interaction between different cell types within the tissue and a fine-tuned transcriptional program synergistically take place to promote beige remodeling. However, both cell-cell interactions and molecular mechanisms governing beige adipocyte appearance and maintenance are poorly understood. In this and the previous issue of Genes & Development, Shao and colleagues (pp. 1461-1474) and Shan and colleagues (pp. 1333-1338) advance our understanding of these issues and, in doing so, highlight potential therapeutic strategies to combat obesity-associated diseases.

ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes.

Energy-storing white adipocytes maintain their identity by suppressing the energy-burning thermogenic gene program of brown and beige adipocytes. Here, we reveal that the protein-protein interaction between the transcriptional coregulator ZFP423 and brown fat determination factor EBF2 is essential for restraining the thermogenic phenotype of white adipose tissue (WAT). Disruption of the ZFP423-EBF2 protein interaction through CRISPR-Cas9 gene editing triggers widespread "browning" of WAT in adult mice. Mechanistically, ZFP423 recruits the NuRD corepressor complex to EBF2-bound thermogenic gene enhancers. Loss of adipocyte Zfp423 induces an EBF2 NuRD-to-BAF coregulator switch and a shift in PPARγ occupancy to thermogenic genes. This shift in PPARγ occupancy increases the antidiabetic efficacy of the PPARγ agonist rosiglitazone in obesity while diminishing the unwanted weight-gaining effect of the drug. These data indicate that ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes and highlight the potential of therapeutically targeting transcriptional brakes to induce beige adipocyte biogenesis in obesity.

Cold-responsive adipocyte progenitors couple adrenergic signaling to immune cell activation to promote beige adipocyte accrual.

The full array of cold-responsive cell types within white adipose tissue that drive thermogenic beige adipocyte biogenesis remains undefined. We demonstrate that acute cold challenge elicits striking transcriptomic changes specifically within DPP4+ PDGFRß+ adipocyte precursor cells, including a ß-adrenergic receptor CREB-mediated induction in the expression of the prothermogenic cytokine, Il33 Doxycycline-inducible deletion of Il33 in PDGFRß+ cells at the onset of cold exposure attenuates ILC2 accumulation and beige adipocyte accrual. These studies highlight the multifaceted roles for adipocyte progenitors and the ability of select mesenchymal subpopulations to relay neuronal signals to tissue-resident immune cells in order to regulate tissue plasticity.

IF1 is a cold-regulated switch of ATP synthase hydrolytic activity to support thermogenesis in brown fat.

While mechanisms controlling uncoupling protein-1 (UCP1) in thermogenic adipocytes play a pivotal role in non-shivering thermogenesis, it remains unclear whether F1Fo-ATP synthase function is also regulated in brown adipose tissue (BAT). Here, we show that inhibitory factor 1 (IF1, encoded by Atp5if1), an inhibitor of ATP synthase hydrolytic activity, is a critical negative regulator of brown adipocyte energy metabolism. In vivo, IF1 levels are diminished in BAT of cold-adapted mice compared to controls. Additionally, the capacity of ATP synthase to generate mitochondrial membrane potential (MMP) through ATP hydrolysis (the so-called "reverse mode" of ATP synthase) is increased in brown fat. In cultured brown adipocytes, IF1 overexpression results in an inability of mitochondria to sustain the MMP upon adrenergic stimulation, leading to a quiescent-like phenotype in brown adipocytes. In mice, adeno-associated virus-mediated IF1 overexpression in BAT suppresses adrenergic-stimulated thermogenesis and decreases mitochondrial respiration in BAT. Taken together, our work identifies downregulation of IF1 upon cold as a critical event for the facilitation of the reverse mode of ATP synthase as well as to enable energetic adaptation of BAT to effectively support non-shivering thermogenesis.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta.

Dermal fibroblasts (dFBs) resist infection by locally differentiating into adipocytes and producing cathelicidin antimicrobial peptide in response to Staphylococcus aureus (S. aureus). Here, we show that neonatal skin was enriched with adipogenic dFBs and immature dermal fat that highly expressed cathelicidin. The pool of adipogenic and antimicrobial dFBs declined after birth, leading to an age-dependent loss of dermal fat and a decrease in adipogenesis and cathelidicin production in response to infection. Transforming growth factor beta (TGF-ß), which acted on uncommitted embryonic and adult dFBs and inhibited their adipogenic and antimicrobial function, was identified as a key upstream regulator of this process. Furthermore, inhibition of the TGF-ß receptor restored the adipogenic and antimicrobial function of dFBs in culture and increased resistance of adult mice to S. aureus infection. These results provide insight into changes that occur in the skin innate immune system between the perinatal and adult periods of life.

The role of ADP-ribose metabolism in metabolic regulation, adipose tissue differentiation, and metabolism.

Poly(ADP-ribose) polymerases (PARPs or ARTDs), originally described as DNA repair factors, have metabolic regulatory roles. PARP1, PARP2, PARP7, PARP10, and PARP14 regulate central and peripheral carbohydrate and lipid metabolism and often channel pathological disruptive metabolic signals. PARP1 and PARP2 are crucial for adipocyte differentiation, including the commitment toward white, brown, or beige adipose tissue lineages, as well as the regulation of lipid accumulation. Through regulating adipocyte function and organismal energy balance, PARPs play a role in obesity and the consequences of obesity. These findings can be translated into humans, as evidenced by studies on identical twins and SNPs affecting PARP activity.

The CD36 scavenger receptor Bez regulates lipid redistribution from fat body to ovaries in Drosophila.

Lipid distribution in an organism is mediated by the interplay between lipoprotein particles, lipoprotein receptors and class B scavenger receptors of the CD36 family. CD36 is a multifunctional protein mediating lipid uptake, mobilization and signaling at the plasma membrane and inside of the cell. The CD36 protein family has 14 members in Drosophila melanogaster, which allows for the differentiated analysis of their functions. Here, we unravel a role for the so far uncharacterized scavenger receptor Bez in lipid export from Drosophila adipocytes. Bez shares the lipid binding residue with CD36 and is expressed at the plasma membrane of the embryonic, larval and adult fat body. Bez loss of function lowers the organismal availability of storage lipids and blocks the maturation of egg chambers in ovaries. We demonstrate that Bez interacts with the APOB homolog Lipophorin at the plasma membrane of adipocytes and trace the Bez-dependent transfer of an alkyne-labeled fatty acid from adipocytes to Lipophorin. Our study demonstrates how lipids are distributed by scavenger receptor-lipoprotein interplay and contribute to the metabolic control of development.

Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism.

Prolonged cold exposure stimulates the recruitment of beige adipocytes within white adipose tissue. Beige adipocytes depend on mitochondrial oxidative phosphorylation to drive thermogenesis. The transcriptional mechanisms that promote remodeling in adipose tissue during the cold are not well understood. Here we demonstrate that the transcriptional coregulator transducin-like enhancer of split 3 (TLE3) inhibits mitochondrial gene expression in beige adipocytes. Conditional deletion of TLE3 in adipocytes promotes mitochondrial oxidative metabolism and increases energy expenditure, thereby improving glucose control. Using chromatin immunoprecipitation and deep sequencing, we found that TLE3 occupies distal enhancers in proximity to nuclear-encoded mitochondrial genes and that many of these binding sites are also enriched for early B-cell factor (EBF) transcription factors. TLE3 interacts with EBF2 and blocks its ability to promote the thermogenic transcriptional program. Collectively, these studies demonstrate that TLE3 regulates thermogenic gene expression in beige adipocytes through inhibition of EBF2 transcriptional activity. Inhibition of TLE3 may provide a novel therapeutic approach for obesity and diabetes.

PGE2 -EP3 axis promotes brown adipose tissue formation through stabilization of WTAP RNA methyltransferase.

Brown adipose tissue (BAT) functions as a thermogenic organ and is negatively associated with cardiometabolic diseases. N6 -methyladenosine (m6 A) modulation regulates the fate of stem cells. Here, we show that the prostaglandin E2 (PGE2 )-E-prostanoid receptor 3 (EP3) axis was activated during mouse interscapular BAT development. Disruption of EP3 impaired the browning process during adipocyte differentiation from pre-adipocytes. Brown adipocyte-specific depletion of EP3 compromised interscapular BAT formation and aggravated high-fat diet-induced obesity and insulin resistance in vivo. Mechanistically, activation of EP3 stabilized the Zfp410 mRNA via WTAP-mediated m6 A modification, while knockdown of Zfp410 abolished the EP3-induced enhancement of brown adipogenesis. EP3 prevented ubiquitin-mediated degradation of WTAP by eliminating PKA-mediated ERK1/2 inhibition during brown adipocyte differentiation. Ablation of WTAP in brown adipocytes abrogated the protective effect of EP3 overexpression in high-fat diet-fed mice. Inhibition of EP3 also retarded human embryonic stem cell differentiation into mature brown adipocytes by reducing the WTAP levels. Thus, a conserved PGE2 -EP3 axis promotes BAT development by stabilizing WTAP/Zfp410 signaling in a PKA/ERK1/2-dependent manner.

The essential role of architectural noncoding RNA Neat1 in cold-induced beige adipocyte differentiation in mice.

Neat1 is an architectural RNA that provides the structural basis for nuclear bodies known as paraspeckles. Although the assembly processes by which Neat1 organizes paraspeckle components are well-documented, the physiological functions of Neat1 are not yet fully understood. This is partly because Neat1 knockout (KO) mice, lacking paraspeckles, do not exhibit overt phenotypes under normal laboratory conditions. During our search for conditions that elicit clear phenotypes in Neat1 KO mice, we discovered that the differentiation of beige adipocytes-inducible thermogenic cells that emerge upon cold exposure-is severely impaired in these mutant mice. Neat1_2, the architectural isoform of Neat1, is transiently upregulated during the early stages of beige adipocyte differentiation, coinciding with increased paraspeckle formation. Genes with altered expression during beige adipocyte differentiation typically cluster at specific chromosomal locations, some of which move closer to paraspeckles upon cold exposure. These observations suggest that paraspeckles might coordinate the regulation of these gene clusters by controlling the activity of certain transcriptional condensates that coregulate multiple genes. We propose that our findings highlight a potential role for Neat1 and paraspeckles in modulating chromosomal organization and gene expression, potentially crucial processes for the differentiation of beige adipocytes.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

Environmental factor reversibly determines cellular identity through opposing Integrators that unify epigenetic and transcriptional pathways.

Organisms must adapt to environmental stresses to ensure their survival and prosperity. Different types of stresses, including thermal, mechanical, and hypoxic stresses, can alter the cellular state that accompanies changes in gene expression but not the cellular identity determined by a chromatin state that remains stable throughout life. Some tissues, such as adipose tissue, demonstrate remarkable plasticity and adaptability in response to environmental cues, enabling reversible cellular identity changes; however, the mechanisms underlying these changes are not well understood. We hypothesized that positive and/or negative "Integrators" sense environmental cues and coordinate the epigenetic and transcriptional pathways required for changes in cellular identity. Adverse environmental factors such as pollution disrupt the coordinated control contributing to disease development. Further research based on this hypothesis will reveal how organisms adapt to fluctuating environmental conditions, such as temperature, extracellular matrix stiffness, oxygen, cytokines, and hormonal cues by changing their cellular identities.

NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis.

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.

p53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Ras/MAPK signaling mediates adipose tissue control of ovarian germline survival and ovulation in Drosophila melanogaster.

From insects to humans, oogenesis is tightly linked to nutritional input, yet little is known about how whole organism physiology matches dietary changes with oocyte development. Considering that diet-induced adipose tissue dysfunction is associated with an increased risk for fertility problems, and other obesity-associated pathophysiologies, it is critical to decipher the cellular and molecular mechanisms linking adipose nutrient sensing to remote control of the ovary and other tissues. Our previous studies in Drosophila melanogaster have shown that amino acid sensing, via the amino acid response pathway and mTOR-mediated signaling function within adipocytes to control germline stem cell maintenance and ovulation, respectively. Additionally, we demonstrated that insulin/insulin-like growth factor signaling within adipocytes employs distinct effector axes, PI3K/Akt1-dependent and -independent, downstream of insulin receptor activity to mediate fat-to-ovary communication. Here, we report that the Ras/MAPK signaling axis functions in adipocytes to regulate early germline cyst survival and ovulation of mature oocytes but is not important for germline stem cell maintenance or the progression through vitellogenesis. Thus, these studies uncover the complexity of signaling pathway activity that mediates inter-organ communication.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism.

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode.

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Comprehensive identification, isolation, and culture of human breast cell types.

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing, and a thorough dissection of the genes and pathways defining each cell type. While the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the 12 major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind-and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

Molecular and cellular mechanisms of chemoresistance in paediatric pre-B cell acute lymphoblastic leukaemia.

Paediatric patients with relapsed B cell acute lymphoblastic leukaemia (B-ALL) have poor prognosis, as relapse-causing clones are often refractory to common chemotherapeutics. While the molecular mechanisms leading to chemoresistance are varied, significant evidence suggests interactions between B-ALL blasts and cells within the bone marrow microenvironment modulate chemotherapy sensitivity. Importantly, bone marrow mesenchymal stem cells (BM-MSCs) and BM adipocytes are known to support B-ALL cells through multiple distinct molecular mechanisms. This review discusses the contribution of integrin-mediated B-ALL/BM-MSC signalling and asparagine supplementation in B-ALL chemoresistance. In addition, the role of adipocytes in sequestering anthracyclines and generating a BM niche favourable for B-ALL survival is explored. Furthermore, this review discusses the role of BM-MSCs and adipocytes in promoting a quiescent and chemoresistant B-ALL phenotype. Novel treatments which target these mechanisms are discussed herein, and are needed to improve dismal outcomes in patients with relapsed/refractory disease.