Adipose-tissue plasticity in health and disease.

Adipose tissue, colloquially known as "fat," is an extraordinarily flexible and heterogeneous organ. While historically viewed as a passive site for energy storage, we now appreciate that adipose tissue regulates many aspects of whole-body physiology, including food intake, maintenance of energy levels, insulin sensitivity, body temperature, and immune responses. A crucial property of adipose tissue is its high degree of plasticity. Physiologic stimuli induce dramatic alterations in adipose-tissue metabolism, structure, and phenotype to meet the needs of the organism. Limitations to this plasticity cause diminished or aberrant responses to physiologic cues and drive the progression of cardiometabolic disease along with other pathological consequences of obesity.

SnapShot: Brown and Beige Adipose Thermogenesis.

Brown and beige adipose tissues have been identified as potential therapeutic targets for combating diet-induced obesity and metabolic disease. Here, we present transcriptional and developmental regulation of brown and beige adipose tissue, as well as critical physiological and pharmaceutical activators of thermogenesis in both tissues.

Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch.

A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here, we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or ß3-agonist treatment. These animals developed obesity on a high-fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the "browning" of white fat and the healthful effects of subcutaneous adipose tissue.

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.

Control of brown and beige fat development.

Brown and beige adipocytes expend chemical energy to produce heat and are therefore important in regulating body temperature and body weight. Brown adipocytes develop in discrete and relatively homogenous depots of brown adipose tissue, whereas beige adipocytes are induced to develop in white adipose tissue in response to certain stimuli - notably, exposure to cold. Fate-mapping analyses have identified progenitor populations that give rise to brown and beige fat cells, and have revealed unanticipated cell-lineage relationships between vascular smooth muscle cells and beige adipocytes, and between skeletal muscle cells and brown fat. In addition, non-adipocyte cells in adipose tissue, including neurons, blood vessel-associated cells and immune cells, have crucial roles in regulating the differentiation and function of brown and beige fat.

EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex.

The transcription factor early B-cell factor 2 (EBF2) is an essential mediator of brown adipocyte commitment and terminal differentiation. However, the mechanisms by which EBF2 regulates chromatin to activate brown fat-specific genes in adipocytes were unknown. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by deep sequencing) analyses in brown adipose tissue showed that EBF2 binds and regulates the activity of lineage-specific enhancers. Mechanistically, EBF2 physically interacts with the chromatin remodeler BRG1 and the BAF chromatin remodeling complex in brown adipocytes. We identified the histone reader protein DPF3 as a brown fat-selective component of the BAF complex that was required for brown fat gene programming and mitochondrial function. Loss of DPF3 in brown adipocytes reduced chromatin accessibility at EBF2-bound enhancers and led to a decrease in basal and catecholamine-stimulated expression of brown fat-selective genes. Notably, Dpf3 is a direct transcriptional target of EBF2 in brown adipocytes, thereby establishing a regulatory module through which EBF2 activates and also recruits DPF3-anchored BAF complexes to chromatin. Together, these results reveal a novel mechanism by which EBF2 cooperates with a tissue-specific chromatin remodeling complex to activate brown fat identity genes.

The tumor suppressor FLCN mediates an alternate mTOR pathway to regulate browning of adipose tissue.

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1ß. Conversely, inducible expression of PGC-1ß in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1ß pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.

Histone deacetylase 3 prepares brown adipose tissue for acute thermogenic challenge.

Brown adipose tissue is a thermogenic organ that dissipates chemical energy as heat to protect animals against hypothermia and to counteract metabolic disease. However, the transcriptional mechanisms that determine the thermogenic capacity of brown adipose tissue before environmental cold are unknown. Here we show that histone deacetylase 3 (HDAC3) is required to activate brown adipose tissue enhancers to ensure thermogenic aptitude. Mice with brown adipose tissue-specific genetic ablation of HDAC3 become severely hypothermic and succumb to acute cold exposure. Uncoupling protein 1 (UCP1) is nearly absent in brown adipose tissue lacking HDAC3, and there is also marked downregulation of mitochondrial oxidative phosphorylation genes resulting in diminished mitochondrial respiration. Remarkably, although HDAC3 acts canonically as a transcriptional corepressor, it functions as a coactivator of oestrogen-related receptor α (ERRα) in brown adipose tissue. HDAC3 coactivation of ERRα is mediated by deacetylation of PGC-1α and is required for the transcription of Ucp1, Ppargc1a (encoding PGC-1α), and oxidative phosphorylation genes. Importantly, HDAC3 promotes the basal transcription of these genes independently of adrenergic stimulation. Thus, HDAC3 uniquely primes Ucp1 and the thermogenic transcriptional program to maintain a critical capacity for thermogenesis in brown adipose tissue that can be rapidly engaged upon exposure to dangerously cold temperature.

PRDM16 binds MED1 and controls chromatin architecture to determine a brown fat transcriptional program.

PR (PRD1-BF1-RIZ1 homologous) domain-containing 16 (PRDM16) drives a brown fat differentiation program, but the mechanisms by which PRDM16 activates brown fat-selective genes have been unclear. Through chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) analyses in brown adipose tissue (BAT), we reveal that PRDM16 binding is highly enriched at a broad set of brown fat-selective genes. Importantly, we found that PRDM16 physically binds to MED1, a component of the Mediator complex, and recruits it to superenhancers at brown fat-selective genes. PRDM16 deficiency in BAT reduces MED1 binding at PRDM16 target sites and causes a fundamental change in chromatin architecture at key brown fat-selective genes. Together, these data indicate that PRDM16 controls chromatin architecture and superenhancer activity in BAT.

Neonatal IL-4 exposure decreases adipogenesis of male rats into adulthood.

The cytokine interleukin 4 (IL-4) can increase beige adipogenesis in adult rodents. However, neonatal animals use a distinct adipocyte precursor compartment for adipogenesis as compared with adults. In this study, we address whether IL-4 can induce persistent effects on adipose tissue when administered subcutaneously in the interscapular region during the neonatal period in Sprague-Dawley rats. We injected IL-4 into neonatal male rats during postnatal days 1-6, followed by analysis of adipose tissue and adipocyte precursors at 2 wk and 10 wk of age. Adipocyte precursors were cultured and subjected to differentiation in vitro. We found that a short and transient IL-4 exposure in neonates upregulated uncoupling protein 1 (Ucp1) mRNA expression and decreased fat cell size in subcutaneous white adipose tissue (WAT). Adipocyte precursors from mature rats that had been treated with IL-4 as neonates displayed a decrease in adiponectin (Adipoq) but no change in Ucp1 expression, as compared with controls. Thus, neonatal IL-4 induces acute beige adipogenesis and decreases adipogenic differentiation capacity long term. Overall, these findings indicate that the neonatal period is critical for adipocyte development and may influence the later onset of obesity.NEW & NOTEWORTHY We used neonatal injections in rat to show that IL-4 decreases adipogenesis and increases browning of white fat. In adulthood, adipocyte precursors show persistently decreased adipogenesis but not increased browning. These studies in the neonate are the first, to our knowledge, to show that IL-4 can have long-lasting effects.

PRDM16 represses the type I interferon response in adipocytes to promote mitochondrial and thermogenic programing.

Brown adipose has the potential to counteract obesity, and thus, identifying signaling pathways that regulate the activity of this tissue is of great clinical interest. PRDM16 is a transcription factor that activates brown fat-specific genes while repressing white fat and muscle-specific genes in adipocytes. Whether PRDM16 also controls other gene programs to regulate adipocyte function was unclear. Here, we identify a novel role for PRDM16 in suppressing type I interferon (IFN)-stimulated genes (ISGs), including Stat1, in adipocytes in vitro and in vivo Ectopic activation of type I IFN signaling in brown adipocytes induces mitochondrial dysfunction and reduces uncoupling protein 1 (UCP1) expression. Prdm16-deficient adipose displays an exaggerated response to type I IFN, including higher STAT1 levels and reduced mitochondrial gene expression. Mechanistically, PRDM16 represses ISGs through binding to promoter regions of these genes and blocking the activating function of IFN regulatory factor 1 (IRF1). Together, these data indicate that PRDM16 diminishes responsiveness to type I IFN in adipose cells to promote thermogenic and mitochondrial function.

Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.

Obesity is an increasingly prevalent disease regulated by genetic and environmental factors. Emerging studies indicate that immune cells, including monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate adaptive immunity and eosinophil and alternatively activated macrophage responses, and were recently identified in murine white adipose tissue (WAT) where they may act to limit the development of obesity. However, ILC2s have not been identified in human adipose tissue, and the mechanisms by which ILC2s regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human WAT and demonstrate that decreased ILC2 responses in WAT are a conserved characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by increasing caloric expenditure. This was associated with recruitment of uncoupling protein 1 (UCP1)(+) beige adipocytes in WAT, a process known as beiging or browning that regulates caloric expenditure. IL-33-induced beiging was dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was sufficient to drive beiging independently of the adaptive immune system, eosinophils or IL-4 receptor signalling. We found that ILC2s produce methionine-enkephalin peptides that can act directly on adipocytes to upregulate Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these studies indicate that, in addition to responding to infection or tissue damage, ILC2s can regulate adipose function and metabolic homeostasis in part via production of enkephalin peptides that elicit beiging.

Deficiency of bone morphogenetic protein-3b induces metabolic syndrome and increases adipogenesis.

Bone morphogenetic protein (BMP) receptor signaling is critical for the regulation of the endocrine system and cardiovascular structure and function. The objective of this study was to investigate whether Bmp3b, a glycoprotein synthetized and secreted by adipose tissue, is necessary to regulate glucose and lipid metabolism, adipogenesis, and cardiovascular remodeling. Over the course of 4 mo, Bmp3b-knockout (Bmp3b-/-) mice gained more weight than wild-type (WT) mice. The plasma levels of cholesterol and triglycerides were higher in Bmp3b-/- mice than in WT mice. Bmp3b-/- mice developed insulin resistance and glucose intolerance. The basal heart rate was higher in Bmp3b-/- mice than in WT mice, and echocardiography revealed eccentric remodeling in Bmp3b-/- mice. The expression of adipogenesis-related genes in white adipose tissue was higher in Bmp3b-/- mice than in WT control mice. In vitro studies showed that Bmp3b modulates the activity of the C/ebpα promoter, an effect mediated by Smad2/3. The results of this study suggest that Bmp3b is necessary for the maintenance of homeostasis in terms of age-related weight gain, glucose metabolism, and left ventricular (LV) remodeling and function. Interventions that increase the level or function of BMP3b may decrease cardiovascular risk and pathological cardiac remodeling.

The nuclear receptor Rev-erbα controls circadian thermogenic plasticity.

Circadian oscillation of body temperature is a basic, evolutionarily conserved feature of mammalian biology. In addition, homeostatic pathways allow organisms to protect their core temperatures in response to cold exposure. However, the mechanism responsible for coordinating daily body temperature rhythm and adaptability to environmental challenges is unknown. Here we show that the nuclear receptor Rev-erbα (also known as Nr1d1), a powerful transcriptional repressor, links circadian and thermogenic networks through the regulation of brown adipose tissue (BAT) function. Mice exposed to cold fare considerably better at 05:00 (Zeitgeber time 22) when Rev-erbα is barely expressed than at 17:00 (Zeitgeber time 10) when Rev-erbα is abundant. Deletion of Rev-erbα markedly improves cold tolerance at 17:00, indicating that overcoming Rev-erbα-dependent repression is a fundamental feature of the thermogenic response to cold. Physiological induction of uncoupling protein 1 (Ucp1) by cold temperatures is preceded by rapid downregulation of Rev-erbα in BAT. Rev-erbα represses Ucp1 in a brown-adipose-cell-autonomous manner and BAT Ucp1 levels are high in Rev-erbα-null mice, even at thermoneutrality. Genetic loss of Rev-erbα also abolishes normal rhythms of body temperature and BAT activity. Thus, Rev-erbα acts as a thermogenic focal point required for establishing and maintaining body temperature rhythm in a manner that is adaptable to environmental demands.

Single-cell transcriptomics and functional target validation of brown adipocytes show their complex roles in metabolic homeostasis.

Brown adipocytes (BAs) are specialized for adaptive thermogenesis and, upon sympathetic stimulation, activate mitochondrial uncoupling protein (UCP)-1 and oxidize fatty acids to generate heat. The capacity for brown adipose tissue (BAT) to protect against obesity and metabolic disease is recognized, yet information about which signals activate BA, besides ß3-adrenergic receptor stimulation, is limited. Using single-cell transcriptomics, we confirmed the presence of mRNAs encoding traditional BAT markers (i.e., UCP1, expressed in 100% of BAs Adrb3, expressed in <50% of BAs) in mouse and have shown single-cell variability (>1000-fold) in their expression at both the mRNA and protein levels. We further identified mRNAs encoding novel markers, orphan GPCRs, and many receptors that bind the classic neurotransmitters, neuropeptides, chemokines, cytokines, and hormones. The transcriptome variability between BAs suggests a much larger range of responsiveness of BAT than previously recognized and that not all BAs function identically. We examined the in vivo functional expression of 12 selected receptors by microinjecting agonists into live mouse BAT and analyzing the metabolic response. In this manner, we expanded the number of known receptors on BAs at least 25-fold, while showing that the expression of classic BA markers is more complex and variable than previously thought.

Ebf2 is a selective marker of brown and beige adipogenic precursor cells.

Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.

Transcriptional control of brown adipocyte development and physiological function--of mice and men.

The last several years have seen an explosion of information relating to the transcriptional control of brown fat cell development. At the same time, new data have emerged that clearly demonstrate that adult humans do indeed have substantial amounts of functioning brown adipose tissue (BAT). Together, these advances are stimulating a reassessment of the role of brown adipose tissue in human physiology and pathophysiology. These data have also opened up exciting new opportunities for the development of entirely novel classes of therapeutics for metabolic diseases like obesity and type 2 diabetes.

Activation of ß3-adrenoceptors increases in vivo free fatty acid uptake and utilization in brown but not white fat depots in high-fat-fed rats.

Activation of brown adipose tissue (BAT) and browning of white adipose tissue (WAT) present potential new therapies for obesity and type 2 diabetes. Here, we examined the effects of ß3-adrenergic stimulation on tissue-specific uptake and storage of free fatty acids (FFA) and its implications for whole body FFA metabolism in diet-induced obese rats using a multi-radiotracer technique. Male Wistar rats were high fat-fed for 12 wk and administered ß3-agonist CL316,243 (CL, 1 mg·kg-1·day-1) or saline via osmotic minipumps during the last 3 wk. The rats were then fasted and acutely infused with a tracer mixture ([14C]palmitate and the partially metabolized R-[3H]bromopalmitate) under anesthesia. CL infusion decreased body weight gain and fasting plasma glucose levels. While core body temperature was unaffected, infrared thermography showed an increase in tail heat dissipation following CL infusion. Interestingly, CL markedly increased both FFA storage and utilization in interscapular and perirenal BAT, whereas the flux of FFA to skeletal muscle was decreased. In this rat model of obesity, only sporadic populations of beige adipocytes were detected in the epididymal WAT depot of CL-infused rats, and there was no change in FFA uptake or utilization in WAT following CL infusion. In summary, ß3-agonism robustly increased FFA flux to BAT coupled with enhanced utilization. Increased BAT activation most likely drove the increased tail heat dissipation to maintain thermostasis. Our results emphasize the quantitative role of brown fat as the functional target of ß3-agonism in obesity.

Genetically altering organismal metabolism by leptin-deficiency benefits a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease that causes death of motor neurons. ALS patients and mouse models of familial ALS display organismal level metabolic dysfunction, which includes increased energy expenditure despite decreased lean mass. The pathophysiological relevance of abnormal energy homeostasis to motor neuron disease remains unclear. Leptin is an adipocyte-derived hormone that regulates whole-animal energy expenditure. Here, we report that placing mutant superoxide dismutase 1 (SOD1) mice in a leptin-deficient background improves energy homeostasis and slows disease progression. Leptin-deficient mutant SOD1 mice possess increased bodyweight and fat mass, as well as decreased energy expenditure. These observations coincide with enhanced survival, improved strength and decreased motor neuron loss. These results suggest that altering whole-body energy metabolism in mutant SOD1 mice can mitigate disease progression. We propose that manipulations that increase fat mass and reduce energy expenditure will be beneficial in the setting of motor neuron disease.