Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

A single-cell atlas of human and mouse white adipose tissue.

White adipose tissue, once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic and heterogenous, and is involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control and host defence1. High-fat feeding and other metabolic stressors cause marked changes in adipose morphology, physiology and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia and type 2 diabetes2. Here we provide detailed cellular atlases of human and mouse subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells, vascular and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease and provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits and cell types in the function of white adipose tissue across species, depots and nutritional conditions.

The kielin/chordin-like protein (KCP) attenuates high-fat diet-induced obesity and metabolic syndrome in mice.

Obesity and its associated complications such as insulin resistance and non-alcoholic fatty liver disease are reaching epidemic proportions. In mice, the TGF-ß superfamily is implicated in the regulation of white and brown adipose tissue differentiation. The kielin/chordin-like protein (KCP) is a secreted regulator of the TGF-ß superfamily pathways that can inhibit both TGF-ß and activin signals while enhancing bone morphogenetic protein (BMP) signaling. However, KCP's effects on metabolism and obesity have not been studied in animal models. Therefore, we examined the effects of KCP loss or gain of function in mice that were maintained on either a regular or a high-fat diet. KCP loss sensitized the mice to obesity and associated complications such as glucose intolerance and adipose tissue inflammation and fibrosis. In contrast, transgenic mice that expressed KCP in the kidney, liver, and adipose tissues were resistant to developing high-fat diet-induced obesity and had significantly reduced white adipose tissue. Moreover, KCP overexpression shifted the pattern of SMAD signaling in vivo, increasing the levels of phospho (P)-SMAD1 and decreasing P-SMAD3. Adipocytes in culture showed a cell-autonomous effect in response to added TGF-ß1 or BMP7. Metabolic profiling indicated increased energy expenditure in KCP-overexpressing mice and reduced expenditure in the KCP mutants with no effect on food intake or activity. These findings demonstrate that shifting the TGF-ß superfamily signaling with a secreted protein can alter the physiology and thermogenic properties of adipose tissue to reduce obesity even when mice are fed a high-fat diet.

Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.

Adipocyte size and fragility and commercial kit costs impose significant limitations on single-cell RNA sequencing of adipose tissue. Accordingly, we developed a workflow to isolate and sample-barcode nuclei from individual adipose tissue samples, integrating flow cytometry for quality control, counting, and precise nuclei pooling for direct loading onto the popular 10× Chromium controller. This approach can eliminate batch confounding, and significantly reduces poor-quality nuclei, ambient RNA contamination, and droplet loading-associated reagent waste, resulting in pronounced improvements in information content and cost efficiency.

Exploring the heterogeneity of white adipose tissue in mouse and man.

Adipose tissue is a heterogeneous organ, comprising cell types, including mature adipocytes, progenitor cells, immune cells, and vascular cells. Here, we discuss the heterogeneity of human and mouse white adipose tissue in general and white adipocytes specifically, focusing on how our understanding of adipocyte subpopulations has expanded with the advent of single nuclear RNA sequencing and spatial transcriptomics. Furthermore, we discuss critical remaining questions regarding how these distinct populations arise, how their functions differ from one another, and which potentially contribute to metabolic pathophysiology.

Hepatic IRF3 fuels dysglycemia in obesity through direct regulation of Ppp2r1b.

Inflammation has profound but poorly understood effects on metabolism, especially in the context of obesity and nonalcoholic fatty liver disease (NAFLD). Here, we report that hepatic interferon regulatory factor 3 (IRF3) is a direct transcriptional regulator of glucose homeostasis through induction of Ppp2r1b, a component of serine/threonine phosphatase PP2A, and subsequent suppression of glucose production. Global ablation of IRF3 in mice on a high-fat diet protected against both steatosis and dysglycemia, whereas hepatocyte-specific loss of IRF3 affects only dysglycemia. Integration of the IRF3-dependent transcriptome and cistrome in mouse hepatocytes identifies Ppp2r1b as a direct IRF3 target responsible for mediating its metabolic actions on glucose homeostasis. IRF3-mediated induction of Ppp2r1b amplified PP2A activity, with subsequent dephosphorylation of AMPKα and AKT. Furthermore, suppression of hepatic Irf3 expression with antisense oligonucleotides reversed obesity-induced insulin resistance and restored glucose homeostasis in obese mice. Obese humans with NAFLD displayed enhanced activation of liver IRF3, with reversion after bariatric surgery. Hepatic PPP2R1B expression correlated with HgbA1C and was elevated in obese humans with impaired fasting glucose. We therefore identify the hepatic IRF3-PPP2R1B axis as a causal link between obesity-induced inflammation and dysglycemia and suggest an approach for limiting the metabolic dysfunction accompanying obesity-associated NAFLD.

Mesothelial cells are not a source of adipocytes in mice.

Visceral adipose tissue (VAT) depots are associated with the adverse metabolic consequences of obesity, such as insulin resistance. The developmental origin of VAT depots and the identity and regulation of adipocyte progenitor cells have been active areas of investigation. In recent years, a paradigm of mesothelial cells as a source of VAT adipocyte progenitor cells has emerged based on lineage tracing studies using the Wilms' tumor gene, Wt1, as a marker for cells of mesothelial origin. Here, we show that Wt1 expression in adipose tissue is not limited to the mesothelium but is also expressed by a distinct preadipocyte population in mice and humans. We identify keratin 19 (Krt19) as a highly specific marker for the adult mouse mesothelium and demonstrate that Krt19-expressing mesothelial cells do not differentiate into visceral adipocytes. These results contradict the assertion that the VAT mesothelium can serve as a source of adipocytes.

Adrenergic-Independent Signaling via CHRNA2 Regulates Beige Fat Activation.

Maintaining energy homeostasis upon environmental challenges, such as cold or excess calorie intake, is essential to the fitness and survival of mammals. Drug discovery efforts targeting ß-adrenergic signaling have not been fruitful after decades of intensive research. We recently identified a new beige fat regulatory pathway mediated via the nicotinic acetylcholine receptor subunit CHRNA2. Here, we generated fat-specific Chrna2 KO mice and observed thermogenic defects in cold and metabolic dysfunction upon dietary challenges caused by adipocyte-autonomous regulation in vivo. We found that CHRNA2 signaling is activated after acute high fat diet feeding and this effect is manifested through both UCP1- and creatine-mediated mechanisms. Furthermore, our data suggested that CHRNA2 signaling may activate glycolytic beige fat, a subpopulation of beige adipocytes mediated by GABPα emerging in the absence of ß-adrenergic signaling. These findings reveal the biological significance of the CHRNA2 pathway in beige fat biogenesis and energy homeostasis.

Development, activation, and therapeutic potential of thermogenic adipocytes.

During the last decade, significant progress has been made in understanding adipocytes with a particular focus on thermogenic fat cells, which effectively convert chemical energy into heat in addition to their other metabolic functions. It has been increasingly recognized that different types and subtypes of adipocytes exist and the developmental origins of various types of fat cells are being intensively investigated. Previous work using immortalized fat cell lines has established an intricate transcriptional network that regulates adipocyte function. Recent work has illustrated how these key transcriptional components mediate thermogenic activation in fat cells. Last but not least, cumulative evidence supports an incontestable role of thermogenic fat in influencing systemic metabolism in humans. Here we summarize the exciting advancements in our understanding of thermogenic fat, discuss the advantages and limitations of the experimental tools currently available, and explore the future directions of this fast-evolving field.

HDAC3-Selective Inhibition Activates Brown and Beige Fat Through PRDM16.

It has been reported that class I histone deacetylase (HDAC) inhibition increases thermogenesis in fat, but adipocyte-specific Hdac3 deletions have presented inconsistent results. In this study, we observed that HDAC3 protein levels were lower in brown fat compared with inguinal subcutaneous adipose tissue, and they decreased in both fat depots upon cold exposure. PR domain-containing 16 (PRDM16) physically interacted with HDAC3, and treatment with HDAC3-selective inhibitor RGFP966 induced thermogenic gene expression in murine and human fat cultures. This induction was blunted in the absence of PRDM16. Our results provide evidence that HDAC3 is involved in thermogenesis, suggesting selective inhibition of HDAC3 in brown and beige fat might hold therapeutic potential for counteracting human obesity and metabolic disorders.

An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling.

Beige adipocytes have recently been shown to regulate energy dissipation when activated and help organisms defend against hypothermia and obesity. Prior reports indicate that beige-like adipocytes exist in adult humans and that they may present novel opportunities to curb the global epidemic in obesity and metabolic illnesses. In an effort to identify unique features of activated beige adipocytes, we found that expression of the cholinergic receptor nicotinic alpha 2 subunit (Chrna2) was induced in subcutaneous fat during the activation of these cells and that acetylcholine-producing immune cells within this tissue regulated this signaling pathway via paracrine mechanisms. CHRNA2 functioned selectively in uncoupling protein 1 (Ucp1)-positive beige adipocytes, increasing thermogenesis through a cAMP- and protein kinase A-dependent pathway. Furthermore, this signaling via CHRNA2 was conserved and present in human subcutaneous adipocytes. Inactivation of Chrna2 in mice compromised the cold-induced thermogenic response selectively in subcutaneous fat and exacerbated high-fat diet-induced obesity and associated metabolic disorders, indicating that even partial loss of beige fat regulation in vivo had detrimental consequences. Our results reveal a beige-selective immune-adipose interaction mediated through CHRNA2 and identify a novel function of nicotinic acetylcholine receptors in energy metabolism. These findings may lead to identification of therapeutic targets to counteract human obesity.

Isolation of Mouse Stromal Vascular Cells for Monolayer Culture.

Positive energy balance contributes to adipose tissue expansion and dysfunction, which accounts largely for obesity and related metabolic disorders. Thermogenic fat can dissipate energy, activation or induction of which may promote energy balance and address the pressing health issues. Recent studies have shown that stromal vascular fraction (SVF) from white adipose tissue (WAT) can develop both white and brown-like adipocyte phenotypes, thus serving as a unique model to study adipogenesis and thermogenesis. Here, we describe a protocol for effective isolation of mouse SVF from WAT, induction of differentiation, and detection of adipogenesis. Success tips for isolation and culture of SVF are also discussed.

Cinnamaldehyde induces fat cell-autonomous thermogenesis and metabolic reprogramming.

OBJECTIVE: Cinnamaldehyde (CA) is a food compound that has previously been observed to be protective against obesity and hyperglycemia in mouse models. In this study, we aimed to elucidate the mechanisms behind this protective effect by assessing the cell-autonomous response of primary adipocytes to CA treatment. METHODS: Primary murine adipocytes were treated with CA and thermogenic and metabolic responses were assessed after both acute and chronic treatments. Human adipose stem cells were differentiated and treated with CA to assess whether the CA-mediated signaling is conserved in humans. RESULTS: CA significantly activated PKA signaling, increased expression levels of thermogenic genes and induced phosphorylation of HSL and PLIN1 in murine primary adipocytes. Inhibition of PKA or p38 MAPK enzymatic activity markedly inhibited the CA-induced thermogenic response. In addition, chronic CA treatment regulates metabolic reprogramming, which was partially diminished in FGF21KO adipocytes. Importantly, both acute and chronic effects of CA were observed in human adipose stem cells isolated from multiple donors of different ethnicities and ages and with a variety of body mass indexes (BMI). CONCLUSIONS: CA activates thermogenic and metabolic responses in mouse and human primary subcutaneous adipocytes in a cell-autonomous manner, giving a mechanistic explanation for the anti-obesity effects of CA observed previously and further supporting its potential metabolic benefits on humans. Given the wide usage of cinnamon in the food industry, the notion that this popular food additive, instead of a drug, may activate thermogenesis, could ultimately lead to therapeutic strategies against obesity that are much better adhered to by participants.

Transcriptional control and hormonal response of thermogenic fat.

Obesity and its associated metabolic diseases present a major public health problem around the world. The discovery that thermogenic fat is active in adult humans has sparked a renewal of interest in the study of its development and function and in the feasibility of using modulators of thermogenesis to work against obesity. In recent years, it has been shown that there are at least two distinct types of thermogenic fat cells: brown and beige fat. In this review, we discuss the transcriptional mediators of thermogenesis and the signaling molecules that regulate thermogenic cells. We also review the effects of thermogenic fat activation on whole-body metabolic parameters and evaluate the increasing evidence that activating thermogenesis in humans can be a viable method of ameliorating obesity. In these discussions, we highlight targets that can potentially be stimulated or modified in anti-obesity treatments.

Using a 3D Culture System to Differentiate Visceral Adipocytes In Vitro.

It has long been recognized that body fat distribution and regional adiposity play a major role in the control of metabolic homeostasis. However, the ability to study and compare the cell autonomous regulation and response of adipocytes from different fat depots has been hampered by the difficulty of inducing preadipocytes isolated from the visceral depot to differentiate into mature adipocytes in culture. Here, we present an easily created 3-dimensional (3D) culture system that can be used to differentiate preadipocytes from the visceral depot as robustly as those from the sc depot. The cells differentiated in these 3D collagen gels are mature adipocytes that retain depot-specific characteristics, as determined by imaging, gene expression, and functional assays. This 3D culture system therefore allows for study of the development and function of adipocytes from both depots in vitro and may ultimately lead to a greater understanding of site-specific functional differences of adipose tissues to metabolic dysregulation.

Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) regulates glucocorticoid action in adipocytes.

Local modulation of glucocorticoid action in adipocytes regulates adiposity and systemic insulin sensitivity. However, the specific cofactors that mediate glucocorticoid receptor (GR) action in adipocytes remain unclear. Here we show that the silencing mediator of retinoid and thyroid hormone receptors (SMRT) is recruited to GR in adipocytes and regulates ligand-dependent GR function. Decreased SMRT expression in adipocytes in vivo increases expression of glucocorticoid-responsive genes. Moreover, adipocytes with decreased SMRT expression exhibit altered glucocorticoid regulation of lipolysis. We conclude that SMRT regulates the metabolic functions of GR in adipocytes in vivo. Modulation of GR-SMRT interactions in adipocytes represents a novel approach to control the local degree of glucocorticoid action and thus influence adipocyte metabolic function.