REvIEWS

MEtabolic SignAlLing Adipogenesis and metabolic health

Alexandra L. Ghaben and Philipp E. Scherer * Abstract | Obesity is characterized by increased adipose mass and has been associated with a strong predisposition towards metabolic diseases and cancer. Thus, it constitutes a public health issue of major proportion. The expansion of adipose depots can be driven either by the increase in size (hypertrophy) or by the formation of new from precursor differentiation in the process of adipogenesis (hyperplasia). Notably , adipocyte expansion through adipogenesis can offset the negative metabolic effects of obesity , and the mechanisms and regulators of this adaptive process are now emerging. Over the past several years, we have learned a considerable amount about how adipocyte fate is determined and how adipogenesis is regulated by signalling and systemic factors. We have also gained appreciation that the adipogenic niche can influence tissue adipogenic capability. Approaches aimed at increasing adipogenesis over adipocyte hypertrophy can now be explored as a means to treat metabolic diseases.

Insulin resistance Overnutrition is currently one of the most costly chal- preadipocytes are -​like in and located 5,6 A pathological condition in lenges for public health. The increasing prevalence of in the perivasculature . which the systemic and cellular obesity sparked great interest in understanding the The balance of hypertrophic expansion of existing response to action is physiological mechanisms promoting effective calorie adipocytes and adipogenesis within an individual has impaired. At the cellular level, storage while minimizing the adverse metabolic conse- a profound impact on metabolic health. As early as insulin resistance can be 7,8 defined as reduced signal quences of obesity such as insulin resistance, dyslipidae- the mid-20th century , increased adipocyte size was transduction along the PI3K– mia, hepatic steatosis, coagulopathies and hypertension. correlated with increased systemic insulin resistance. AKT–mTOR pathway per unit Although the role of adipocytes in energy storage is Additional studies have suggested that small adipocytes of insulin, resulting in firmly established, we have only recently begun to are especially important for counteracting obesity-​ decreased glucose uptake per 9,10 unit of insulin in adipocytes appreciate the profound influence of adipocytes on other associated metabolic decline and have shown that and skeletal muscle or aspects of systemic metabolic homeostasis. Indeed, the small adipocytes are often correlated with decreased decreased suppression of ability to sequester lipid effectively inside adipocytes susceptibility to developing diabetes11,12. As they expand hepatic gluconeogenesis. prevents toxic lipid accumulation (lipotoxicity) in other in size, adipocytes experience increased mechanical Systemically, compared with tissues, such as muscle, liver and heart, and firmly cor- stress as their contact with neighbouring cells and healthy individuals, individuals with insulin resistance have relates with preserved metabolic function at all levels extracellular components increases, and they elevated plasma insulin levels, of pathology associated with obesity. The question experience hypoxia as they expand to sizes approaching elevated plasma glucose levels remains: what factors govern the expansion of adipose the limits of oxygen diffusion. The increased mechan- and impaired glucose depots and how are they regulated? ical and hypoxic stresses of hypertrophic adipocytes clearance in response to the 13,14 same bolus of insulin. can increase in size in one of two main contribute to adipose tissue inflammation . Many ways: hypertrophy (increase in size of existing adipo- experimental observations suggest that larger, hyper- cytes) or hyperplasia (formation of new adipocytes trophic adipocytes may display different biochemical through differentiation of resident precursors known properties than smaller adipocytes do. These differ- as preadipocytes) (Fig. 1). Differentiated adipocytes ences include elevated lipolysis15, increased inflam- have remarkable hypertrophic potential, being able matory cytokine secretion16 and reduced secretion to increase in size to several hundred micrometres in of anti-​inflammatory adipokines such as leptin16 and Touchstone Diabetes Center, diameter. The number of adipocytes in a given depot is adiponectin17,18. Department of Internal primarily determined early in life and is mostly stable Here, we outline the cellular, tissue-​specific and Medicine, The University of through adulthood1,2. However, recent lineage-​tracing systemic mechanisms influencing adipogenesis. We Texas Southwestern Medical Center, Dallas, TX, USA. models in rodents have demonstrated that, during pro- also discuss the ways in which adipocytes influence *e-mail:​ philipp.scherer@ longed caloric excess, new adipocytes can emerge from physiology and discuss how adult adipogenesis (or lack UTSouthwestern.edu the differentiation of preadipocytes and can contribute thereof) alters the systemic metabolic state, highlighting 3,4 https://doi.org/10.1038/ to adipose tissue expansion . Generally, these obser- the beneficial role of adipogenesis in metabolic health s41580-018-0093-z vations have led to the appreciation that these adult and fitness.

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Beneficial adipokines • FGF21: stimulating hepatic gluconeogenesis • : suppressing food intake, promoting lipolysis • : insulin sensitizing, suppressing hepatic glucose output, anti-inflammatory and anti-fibrotic

M Angiogenesis G2 G1 Expenditure Healthy Metabolically • Basal metabolism storage S healthy • Physical activity obesity • Thermogenesis Hyperplasia Perivascular Adipocytes Intake preadipocyte • High Increased levels of • High sugar glucose and lipids Hypoxia Necrosis in circulation Nutrient and calorie excess Ectopic lipid deposition Adipose tissue Metabolically Unhealthy unhealthy dysfunction storage Hypertrophy obesity Inflammation

Fig. 1 | Mechanisms of adipose tissue expansion. Storage of extra calories in adipose tissue is required in states of overnutrition, when caloric excess exceeds energy expenditure. Adipose tissue can primarily expand in one of two ways: through differentiation of resident tissue precursors to form new adipocytes (hyperplasia) or through enlargement of existing adipocytes (hypertrophy). Hyperplasia of adipose tissue is generally considered healthy and adaptive, because the tissue is able to maintain proper vascularization and levels of the insulin-sensitizing,​ anti-inflammatory​ adiponectin and other metabolism-modulatory​ adipokines. Hypertrophy of adipocytes is associated with an increase in hypoxia experienced by these cells because of their massively expanded size. Unlike in most other tissues, however, the hypoxic response of adipose tissue (mediated through hypoxia-inducible​ factor 1) is insufficient to induce vascularization. Instead, hypoxic adipose tissue increases the expression of pro-fibrotic​ genes and leads to tissue fibrosis. Sometimes, hypoxic adipocytes undergo necrosis, leading to infiltration by immune cells and tissue inflammation. These factors combine to decrease adipose tissue function, leading to persistently elevated levels of nutrients (sugars and lipids) in the blood and contributing to earlier onset of metabolic disease and causing toxic lipid deposition in other tissues, such as muscle and liver.

Physiological roles of white adipocytes such as hormone-​sensitive lipase (HSL) and adipose The defining feature of adipocytes is their ability to store triglyceride lipase (ATGL; also known as PNPLA2) to excess calories in the form of triglycerides, which are release non-​esterified fatty acids and glycerol from the Lipotoxicity packaged into large lipid droplets that take up most of lipid droplets into circulation for use by organs, such as A cellular dysfunction arising the cell. Storing energy as triglycerides is evolutionar- the liver and skeletal muscle. In times of nutrient excess, from accumulation of lipid ily advantageous; however, the presence of adipocytes, blood glucose levels rise, and the pancreatic hormone intermediates in cells other than adipocytes. In liver, which are specialized for this purpose, is limited to verte­ insulin suppresses HSL and ATGL and promotes glu- accumulation of lipids brates. In addition to their energy-​storing role, adipo- cose uptake and de novo lipogenesis to package lipids contributes to the cytes provide several additional physiological functions. into lipid droplets and store the excess nutrients. During pathogenesis of non-alcoholic​ Animals in cold climates have increased adiposity, which obesity-associated​ metabolic decline, the adipocyte fails fatty liver disease and to insulin resistance. Lipid accumulation endows them with increased insulation and thermogen- to respond properly to almost all these extracellular sig- in skeletal muscle can esis. Adipocytes also provide mechanical cushioning and nals, most notably insulin, leading to elevated plasma contribute to insulin resistance, are abundant in anatomical regions experiencing high glucose and lipids (reviewed elsewhere23,24). There are whereas in cardiac muscle, it mechanical stress such as the palm, buttocks and heel. additional characteristics of adipocyte dysfunction dur- can cause apoptotic cell death. Internally, adipose tissue provides cushioning to organs ing obesity-​associated metabolic decline, including an Pancreatic lipid accumulation can lead to dysregulation of such as the heart, adrenal glands, kidneys and ovaries. altered transcriptional programme with an increased β-cell​ insulin secretion, and Additionally, there is a second class of adipocytes found rate of collagen synthesis and increased rates of adipo- ultimately to apoptotic cell in euthermic mammals, referred to as brown or beige cyte necrosis, which are associated with increased levels death. adipocytes, that are specialized in energy and heat dissi- of pro-​inflammatory cytokines25. (Box 1) Adipokines pation . For the purposes of this Review, we focus The past 20 years have expanded the role of adipose Signalling molecules (proteins on white adipocytes, and the reader is referred to other tissue to include its dynamic function as an endocrine or lipids) secreted from articles focused on brown and beige adipogenesis19–22. organ. Initiated by the discovery of leptin26, followed adipose tissue. Classically, adipocytes respond to systemic signals closely by adiponectin27, we now appreciate that white to either store or mobilize nutrients as needed by the adipose tissue secretes many signalling proteins, known Euthermic A term referring to an animal organism. In times of nutrient deprivation, blood glu- as adipokines. Many of these adipokines help to coordin­ that maintains a constant body cose drops and adipocytes are stimulated by ate the systemic metabolic state. For example, adipo­ temperature. such as or noradrenaline to activate lipases, nectin suppresses hepatic glucose output and reduces

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16 Interscapular depot fibrosis and inflammation in many other tissues; leptin obesity-​associated metabolic decline . By contrast, adi- The acts on the satiety centres in the brain to suppress food pose tissue expansion through enhanced adipogenesis depot found between the intake and increase sympathetic output to promote lipo­ not only distributes excess calories among many smaller, scapulae in rodents and infant lysis; and FGF21 stimulates hepatic gluconeogenesis28,29. newly formed adipocytes but also reduces the number humans. Certain adipokines are also hallmarks of metabolically of hypertrophic adipocytes secreting pro-inflammatory​ Supraclavicular depot dysfunctional adipose tissue and can promote systemic factors. Large adipocytes also experience hypoxic stress The thermogenic adipose inflammation (tumour necrosis factor (TNF), inter- and stimulate collagen deposition and fibrosis of the adi- tissue depot found above the leukin 6 (IL-6) and angiotensin II (ANG II)), insulin pose depots, augmenting inflammatory infiltration and clavicles in adult humans and resistance (adipocyte fatty acid-​binding protein (AP2; pro-inflammatory​ output of the tissue13. The creation of mice, which in humans contains the highest proportion, by also known as FABP4) and progranulin) and athero- smaller adipocytes through adipogenesis correlates with 28,29 volume, of total brown adipose genesis (adipsin and leptin) . There is also growing increased angiogenesis, reducing the hypoxic stress on tissue. evidence that certain lipids secreted from adipocytes can the adipose depot and the subsequent inflammation36. have systemic consequences, including the interesting These results suggest a model in which adipogenesis and Sympathetic output 30 The sympathetic nervous but controversial insulin-sensitizing​ fatty-acid​ esters of the coordinated vascularization of the adipose depot 31 32 system is a system of hydroxyl fatty acids (FAHFAs) and palmitoleate , as allow for preserved function of in peripheral nerves that signal well as pro-angiogenic​ monobutyrin33. By contrast, some obesity (Fig. 1). primarily with the lipid subtypes secreted from the adipose tissue, includ- neurotransmitter noradrenaline ing ceramides, can have potent insulin-​desensitizing Molecular mechanisms of adipogenesis through activation of adrenergic receptors. activities and may contribute to the insulin resistance Adipogenesis is the process whereby fibroblast-​like pro- 34,35 Sympathetic output refers to associated with obesity . genitor cells (Box 2) restrict their fate to the adipogenic central signals that increase Larger adipocytes have been found to correlate with a lineage, accumulate nutrients and become triglyceride-​ the activity of these nerves. more pro-inflammatory​ adipokine profile, exacerbating filled mature adipocytes. We can think of this as a two-​step process. In the first step, a fibroblast-​like cell (characterized by the expression of platelet-​derived Box 1 | Brown and beige adipogenesis growth factor -​α (PDGFRα) and/or PDGFRβ; in contrast to the energy-​storing white adipocyte, brown and beige adipocytes are Box 2), such as a mesenchymal precursor, restricts itself characterized by the capability for energy expenditure through thermogenesis. to the adipocyte lineage without any morphologi- Characteristically, these adipocytes have an increased number of mitochondria and many cal changes, forming a preadipocyte (commitment small lipid droplets, and they express high levels of uncoupling protein 1 (uCP1), which is step). This commitment is then followed by differen- considered the key driver of the thermogenesis programme. in mice, brown and beige tiation, during which specified preadipocytes undergo adipocytes can be distinguished with high confidence. Brown adipocytes develop from an growth arrest, accumulate lipids and form functional, MYF5+ lineage, whereas beige adipocytes do not. Brown adipocytes exist in anatomically (Fig. 2) distinct depots (most notably, the interscapular depot), whereas beige adipocytes are insulin-​responsive mature adipocytes . Much of found in white depots (most notably in subcutaneous depots). Human infants have an our knowledge regarding the molecular mechanisms interscapular brown adipose depot, whereas adult humans lose this depot and instead can of adipogenesis comes from a number of cell culture develop thermogenic adipose tissue in the paraspinal depot and supraclavicular depot models involving fibroblast-​like cells that differentiate with adipocytes molecularly similar to both brown and beige adipocytes. to form adipocytes in response to a hormonal cocktail as discussed, there exists a degree of lineage plasticity between white and beige (see Supplementary Box 1). Applying this knowledge of preadipocytes, although there are several hallmarks that distinguish them. Lineage in vitro adipocyte differentiation to identify naive adipo- tracing in vivo reveals that the beige preadipocytes, like white preadipocytes, are cyte precursors in vivo has not proved straightforward. 235,236 perivascular cells. the brown and beige specific eBF2 (refs ) There is no single consensus marker of preadipocytes recruits peroxisome proliferator-​activated receptor-​ (PPar ) to thermogenic gene γ γ in vivo, likely reflecting the heterogeneity of preadipo­ targets and is necessary for the adipogenesis of beige adipocytes after 3 adrenergic β Box 2 stimulation (an experimental surrogate of cold exposure). it is also sufficient to drive cytes ( ; see also below). Separately, many early adipocyte precursors isolated from white adipose tissue to differentiate into beige inducible lineage-​tracing studies of adipogenesis uti- adipocytes. the white adipocyte commitment factor ZFP423 directly represses eBF2 lized the oestrogen receptor agonist tamoxifen, which transcriptional activity to allow preadipocytes to form classically white adipocytes237. has been shown to act as a lipodystrophic agent that the gonadal depot of mouse adipose tissue is relatively resistant to beige adipogenesis, stimulates de novo adipogenesis and hence interferes and the expansion of the beige adipose tissue preferentially occurs subcutaneously111,238. with endogenous adipogenic programmes37. Below, However, deletion of ZFP423 in preadipocytes ‘unlocks’ beige adipogenesis in the we highlight the main molecular players involved in gonadal depot46. Little is known about the molecular and cellular regulation determining adipogenesis that are known to date. whether a precursor forms a white or beige adipocyte, but some data suggest that morphogenetic protein (BMP) signalling may disrupt the ZFP423–eBF2 interaction, Early adipocyte commitment factors. Adipogenic com- thereby promoting the beige adipocyte fate237. Many molecular inputs are described to influence thermogenic adipocyte mitment is the process whereby a multipotent precursor differentiation and activation (reviewed extensively elswhere21,22), but we briefly draw becomes restricted to the adipocyte lineage and inca- attention to the role of ageing. Notably, diabetes and metabolic disease show high pable of forming other mesenchymal cell types, such incidence in the aged population and are accompanied by a considerable decrease in as myoblasts, chondroblasts or (reviewed basal and cold-recruited​ brown adipocyte activity in vivo. In addition, human elsewhere38–40). preadipocytes from elderly individuals show decreased beige adipogenesis in vitro239. Early studies identified that bone morphogenetic this decline in beige adipogenesis has been linked to and could be protein 2 (BMP2) and BMP4 are sufficient to drive counteracted by overexpression of the NaD-dependent​ histone deacetylase sirt1, adipocyte commitment in cultured and are 239 which counteracts cellular senescence . ageing-induced​ cellular senescence also required for in vitro adipogenic differentiation41–43. appears to diminish beige adipocyte differentiation in vivo, as inhibition of cellular BMPs bind and signal through BMP receptors to acti- senescence in mouse preadipocytes restored beige adipocyte differentiation213. vate the SMAD4 transcription factor by activating its

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Gluconeogenesis heterodimeric partners SMAD1, SMAD5 and SMAD8 implicated in the regulation of adipogenesis. For example, 42 The generation of glucose from (ref. ). The activated SMAD4 is then able to promote ZFP467 suppresses osteogenesis and promotes adipo­ non-carbohydrate​ carbon terminal differentiation by stimulating transcription of genesis of the fibroblast-​like progenitors by enhancing sources (glycerol, odd-​chain peroxisome proliferator-​activated receptor-​γ (PPARγ), the expression of another master regulator of adipogenic fatty acids, lactate or certain amino acids). In mammals, the the master regulator of adipogenesis. gene transcription, co-​activator CCAAT/enhancer- 47 liver, kidney, intestine and Separately, a screen for factors enriched in adipogenic binding protein-α​ (C/EBPα) . Furthermore, KLF5 acts as skeletal muscle are the only cell lines revealed the transcription factor ZFP423 as a co-activator of C/EBPβ, which then enhances PPARγ organs capable of preferentially enriched in adipogenic fibroblasts, where and C/EBPα transcription48. By contrast, GATA2 and gluconeogenesis. it acts to sensitize fibroblasts to pro-​adipogenic BMP GATA3 inhibit adipogenesis through prevention of signalling44. ZFP423 is required for fetal development PPARγ transcription49. Finally, EGR1 and EGR2 inhibit of subcutaneous adipose tissue45 but is not essential for and stimulate adipogenesis, respectively, by modulating visceral adipogenesis during high-​ feeding (see the the activation of cAMP-response element-binding protein following section for a discussion of adipogenic depots), (CREB), which regulates transcription of C/EBPβ50. suggesting other unknown factors can compensate for its It is now very important to gain a better understanding of absence46. Other zinc-​finger transcription proteins are which factors (systemic and intracellular) influence the expression and activity of these different commitment Box 2 | identity of the white adipocyte precursor factors and how these factors are regulated on a molec- ular level (for example, through post-transcriptional and until recently, the predominant source of our knowledge regarding adipocyte post-translational​ mechanisms). differentiation derived from various in vitro mixed-cell-type​ populations including mouse embryonic fibroblasts, the stromal vascular fraction of adipose tissue and the Adipocyte differentiation. The nuclear hormone receptor 3t3-L1 fibroblast-like​ cell line (see supplementary Box 1). However, it has remained unclear whether these in vitro models mimic relevant in vivo precursors and what the PPARγ was initially identified as a transcription factor in vivo characteristics of adipose precursors are. regulating the expression of the adipose-​specific gene One of the broadest markers of the adipocyte lineage is platelet-derived​ growth AP2. PPARγ is characterized as the master regulator of factor α (PDGFrα), and the overwhelming majority of adult adipocytes derive from this adipogenesis, because it is indispensable for adipocyte lineage240. However, PDGFrα is not expressed in mature adipocytes, reflecting that its differentiation in culture51 and in vivo52–54. Numerous expression is restricted to precursors. within adipose depots, PDGFrα+ cells reside near lipid metabolites are proposed as endogenous ligands the vasculature107. alternative approaches attempted to characterize the resident of PPARγ, including the polyunsaturated fatty acids, adipocyte precursor by identifying characteristics of fibroblasts expressing peroxisome eicosanoids and prostaglandins. However, the affinity or + proliferator-activated​ receptor-γ​ (PParγ). Nearly all proliferating PParγ cells were abundance of these species in adipose tissue is low, and localized to the blood vessel walls of adipose depots but not in other tissues5. these the physiological ligand of PPARγ is still unknown55. cells resemble mural cells (also known as pericytes or vascular smooth muscle cells) and Members of the synthetic thiazolidinedione class of express common mural markers, such as α-smooth​ muscle actin (αsMa) and PDGFrβ. independently, screening identified the zinc-​finger protease ZFP423 as a key drugs (TZDs, also called glitazones) are potent PPARγ commitment factor of the white adipocyte linage44, and in vivo tracing localized these ligands that stimulate adipogenesis in vitro and in vivo. highly adipogenic cells to the perivascular mural compartment6. Several in vivo, However, although TZDs are generally considered safe, lineage-​tracing studies confirm that perivascular αsMa+ cells74 and PDGFrβ+ cells some controversial reports of cardiac side effects of these contribute to adipogenesis in mouse models during high-fat-diet​ feeding4. drugs and the weight gain associated with their use have while these perivascular lineage markers, including αsMa, PDGFrβ and PDGFrα, all limited the enthusiasm for their clinical application as mark the adipocyte lineage, they also mark many other cell types throughout the body, pro-adipogenic​ compounds56–58. and thus their utility for identifying preadipocytes is limited. various combinations of One of the most important downstream effects cell surface markers can enrich for preadipocytes (reviewed elsewhere241), but we still of PPARγ is the activation of the transcription factor lack a reliable means of identifying adipogenic precursors at the exclusion of other cell 59 types. several independent groups recently addressed this challenge using unbiased C/EBPα . In cultured fibroblasts, overexpression of 60 single-cell​ sequencing to identify adipogenic populations of stromal cells109,110,160. C/EBPα is sufficient to drive adipocyte differentiation , 61 although each group used a slightly different starting population of cells, all groups although this induction requires PPARγ . To fully described the existence of a molecularly distinct population of cells enriched for activate the mature adipocyte programme, C/EBPα adipogenic genes. in one case, only this adipogenic-gene-enriched​ population and PPARγ functionally synergize. More than 90% underwent spontaneous adipogenesis in vitro, demonstrating functional heterogeneity of PPARγ DNA-​binding sites also bind C/EBPα, and corresponding to transcriptional heterogeneity160. Consistent with the heterogeneous PPARγ requires induction of C/EBP family proteins to development and function of different adipose tissue depots, it appears that the fully activate transcription of genes expressed in mature molecular characteristics of adipogenic cells differ between subcutaneous and visceral adipocytes, such as those encoding the insulin receptor, depots, highlighting the need for increasingly selective markers of preadipocytes110,160. AP2 and adiponectin62. Embryonic adipogenesis is not Future work will address the unresolved questions of the in vivo functional importance 63 and lineage restriction of these populations of stromal cells. dependent on C/EBPα specifically , likely because the Finally, adipocyte differentiation is not a unidirectional process, and certain stimuli family member C/EBPβ shares some functional redun- can prompt adipocytes to return to a phenotypically fibroblast-like​ precursor state. dancy with C/EBPα. Interestingly, this redundancy is Lineage tracing supports this de-differentiation​ in physiological processes such as limited to the embryo, as C/EBPα is required for all wound healing64, tumour growth66,67 and lactation68. in the case of wound healing and forms of adult adipogenesis63. lactation, it appears that some adipocytes may pass reversibly through a precursor stage many times while always retaining the capacity to form a mature adipocyte. Adipocyte de-​differentiation. Adipogenesis has long these fascinating demonstrations of adipocyte plasticity call into question the notion been viewed as a unidirectional process, but new data are of the adipocyte as a terminally differentiated cell type. it would also be interesting to leading us to question whether adipocytes are truly ter- study whether these fibroblast-like​ cells derived from an adipocyte lineage are minally differentiated. A mounting body of data suggests restricted to form only adipocytes or gain pluripotency as a result of de-differentiation.​ that adipocytes retain the capacity to de-​differentiate,

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De-differentiation

Multipotent Committed Mature mesenchymal preadipocyte Differentiating adipocyte preadipocyte precursor BMP4 (ZFP423, (αSMA, PDGFRα/PDGFRβ) PDGFRα/PDGFRβ, αSMA)

Commitment Growth arrest

Early differentiation Mature adipocyte markers markers • PPARγ • Adiponectin, • C/EBPα or C/EBPβ leptin Myoblast Chondroblast • AP2 • ATGL, LPL • GLUT4 • Perilipin

Fig. 2 | overview of molecular mechanisms of adipogenesis. Multipotent, fibroblast-like​ mesenchymal precursors (marked by α-smooth​ muscle actin (αSMA) and platelet-​derived growth factor receptors (PDGFRα and/or PDGFRβ)) serve as adipocyte precursors but are also capable of forming myoblasts, osteoblasts and chondroblasts. Bone morphogenetic protein (BMP) signalling restricts these mesenchymal precursors to the adipocyte lineage as part of a process known as ‘commitment’. At the time of commitment, the precursors are still morphologically fibroblast-like​ and importantly express the transcription factor ZFP423. When the committed preadipocyte arrests its growth, it activates the master regulator of adipogenesis peroxisome proliferator-activated​ receptor-γ​ (PPARγ) and transcription co-activators​ CCAAT/enhancer-​ binding protein α and β (C/EBPα and C/EBPβ). Lipid accumulation drives the expression of the adipocyte fatty-acid​ binding protein (AP2) and the insulin-sensitive​ transporter GLUT4, marking adipocytes in early stages of differentiation. At the completion of differentiation, mature adipocytes express all the markers of early adipocyte differentiation as well as the peptide hormones adiponectin and leptin; the lipases adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL); and high levels of the lipid-droplet-associated​ protein perilipin 1. There is now evidence that adipocytes can undergo de-​differentiation back to fibroblast-like​ preadipocytes. Whether these preadipocytes are committed to the adipocyte lineage or whether they can generate other mesenchymal cell types is unknown.

and it has been proposed that they are able to return to novel avenues for anticancer therapies and potentially a fibroblast-like​ precursor state. Lineage-tracing​ studies also for controlling the number of adipocytes to promote in animal models suggest that fibroblasts within healing weight loss. cutaneous lesions64, fibrotic dermis65, tumour stroma66,67 and lactating mammary glands68 derive from an adipo- Sites of adipogenesis genic lineage. In the case of wound healing and lacta- It is helpful to consider adipose tissue as divided into Ceramides tion, the adipocyte de-​differentiation is reversible, and subcutaneous (localized beneath the ) and visceral A class of sphingolipids extensively reported to the fibroblastic cells resulting from the de-differentiation​ (distributed in the abdominal cavity) depots or fat pads. contribute to insulin resistance. of adipocytes are capable of reforming adipocytes. It is In humans, the predominant subcutaneous depot is The anti-diabetic​ adipokine unclear to what degree these fibroblastic adipocyte the gluteofemoral depot, localized around the hips and adiponectin exhibits effects precursors are restricted to the adipocyte lineage and thighs. The predominant human visceral depot is the partially through receptor-​ whether they are capable of forming other cell types omental depot, which is a large peritoneal fold connect- associated ceramidase activity. in vivo. We also know very little about what (if any) ing the stomach and other visceral organs. In rodent Mesenchymal precursor functional purpose this de-​differentiation event serves. models, the inguinal adipose depot (around the groin) A stromal, fibroblast-like​ cell For example, multiple studies have shown that adipose and the epididymal (or gonadal) adipose depot are the found throughout the body tissue shrinking during weight loss does not decrease predominant models of subcutaneous and visceral adi- that is capable of differentiating to form an adipocyte cell number — at least not within the accu- pose tissue, respectively. In this Review, we continue adipocyte, or racy of the methodologies used, which are not accurate to use subcutaneous and visceral as shorthand to refer to osteoblast. enough to detect alterations within a 10–20% change in the broad categories of adipose tissue but emphasize cell numbers1,69 — and therefore, de-​differentiation of that important differences in depot function exist within Nuclear hormone receptor adipocytes is not driving cell number control in the adi- these categorizations (reviewed elsewhere72). Adipocytes A class of nuclear receptors capable of directly binding to pose tissue, at least not in response to changes in calorie are also physiologically or pathologically associated with both DNA and a steroid or balance. Nevertheless, understanding of the mechanisms nearly every non-neuronal​ organ in the body, including other endocrine hormone. controlling the reversibility of adipose differentiation the skin, liver, , skeletal muscle, vasculature, Nuclear hormone receptors are may provide key insights into certain malignancies. intestine, adrenal glands, pericardium and ovaries. able to directly regulate transcription upon ligand Liposarcomas are malignant tumours resembling adipo- Most adipocytes emerge during development, and binding. cytes and adipose precursors, with malignancy inversely their emergence is tightly associated with blood vessels. correlated with the prevalence of well-​differentiated Increases in adipose tissue in adults are mostly driven by Eicosanoids adipo­cytes in the tumour tissue70. Other types of cancer, adipocyte hypertrophy, but hyperplasia can be induced Straight-chain,​ 20-carbon such as haematomas and haemangiomas, also contain in some circumstances. Notably, the propensity to polyunsaturated fatty acids 71 that contain an oxygen moiety. adipocytes at various states of differentiation . Thus, a generate new adipocytes varies between the different Characteristically, eicosanoids greater understanding of what molecular signals pro- adipogenic sites, and these processes are differentially are signalling lipids. mote or sustain adipose de-​differentiation can provide regulated and have different consequences (Fig. 3).

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Muscle Induction: overnutrition, trauma, muscular dystrophy Preadipocyte: PDGFRα+ Signals: Hedgehog signalling (inhibition)

Subcutaneous adipose depots Bone marrow Induction: cold exposure Induction: anorexia, radiation, Preadipocyte: PDGFRα+ ageing Signals: unclarified Preadipocyte: LepR+ Signals: leptin, Hedgehog Myofibroblast signalling (inhibition) Dermal adipose tissue Induction: wound healing, follicle cycling (telogen Visceral adipose deposits to anagen transition) Induction: overnutrition + + Preadipocyte: Pre-adipocyte: PDGFRα /PDGFRβ • PDGFRα+ and/or ZFP423+ Signals: insulin, • De-differentiation of myofibroblasts Signals: WNT (inhibition), Adipocyte NOTCH (de-differentiation)

Fig. 3 | Sites of adult adipogenesis. Adipocytes are found throughout the adult body. The main two niches are the subcutaneous and visceral fat depots. Other adipogenic niches include the , bone marrow and skeletal muscle. Notably , every niche delivers unique stimuli controlling adipogenesis and harbours unique preadipocyte populations with different markers and/or adipogenic capabilities. Platelet-derived​ growth factor receptor-α​ expressing (PDGFRα+) preadipocytes in subcutaneous adipose tissue normally do not expand by hyperplasia but are stimulated to form beige adipocytes by cold temperatures. Hyperplastic growth can be induced in the visceral adipose tissue depot. During high-​ fat feeding, PDGFRα+ and/or PDGFRβ+ preadipocytes in the visceral depot are stimulated by insulin and glucocorticoids to create additional adipocytes for nutrient storage. Dermal preadipocytes arise from a PDGFRα+ and/or ZFP423+ lineage; they proliferate and generate new adipocytes as part of hair follicle cycling and cutaneous wound healing. Activation of WNT–β-catenin​ signalling in dermal preadipocytes inhibits their differentiation, whereas NOTCH activation drives adipocyte de-differentiation,​ leading to the depletion of dermal adipocytes. After muscular injury or disease (such as muscular dystrophies) or during severe insulin resistance and increased levels of glucose, adipocyte differentiation is also induced in the muscle, where adipogenesis is normally restricted by Hedgehog signalling. In bone marrow , adipogenesis often occurs after injury , such as radiation or fracture, and is also associated with ageing and anorexia. In this depot, adipogenesis is induced by leptin, and adipogenic precursors are leptin-receptor-positive​ (LepR+).

Fetal adipogenesis. In humans, adipose tissue appears interpretation of mouse models developed with this strat­ early in the second trimester of pregnancy in a cranial egy is then required, as adipogenesis and development to caudal and then medial to lateral fashion73. In mice, of the adipose depots can be altered in these mice. subcutaneous adipose is formed embryonically, whereas Physiologically, the advanced state of differentiation the visceral depots develop during the first 2 postnatal of fetal precursors suggests that these cells are primed weeks3. Evidence is mounting that fetal and adult adipo­ to respond to external and internal cues of adipogenesis genesis involves different precursor populations and differently. It is easy to imagine factors such as maternal distinct regulatory processes. Early lineage-tracing​ stud- nutrition state, that could influence the developmental Prostaglandins ies provide data to suggest that adult and fetal precursors patterning of adipose tissue, with lifelong implications A subclass of eicosanoids are distinctly defined populations74. Unlike in adults, for the organism. derived from arachidonic acid fetal adipogenesis appears to be surprisingly C/EBPα with a characteristic five-​ 63 (ref.45) Visceral and subcutaneous white adipose tissue carbon ring. The signalling independent but requires ZFP423 . Evidence actions of prostaglandins are also suggests that the perivascular phenotype of adi- depots in adults. The bulk of adipose mass in adults is highly tissue-specific.​ pose precursors is adopted postnatally, whereas fetal present within the broadly defined subcutaneous and precursors are found throughout the developing stroma visceral depots demarcated by the peritoneum. These Gluteofemoral depot of adipose depots74. Additionally, fetal precursors might two classes of adipose depots have intrinsically different A representative, commonly studied human subcutaneous be at a slightly more differentiated state than their adult metabolic functions, including secretion of adipokines adipose depot. This depot is counterparts, evidenced by their expression of the lipid and inflammatory cytokines, lipolysis rates and thermo- anatomically located droplet protein perilipin and the adipokine adiponectin, genic potential. Numerous human and rodent studies subcutaneously, along the hips, the expression of which is conventionally restricted to correlate increased subcutaneous adiposity relative to buttocks and thighs. mature adipocytes in adult animals75. visceral adiposity with preserved metabolic health for 72 Omental depot The difference in the molecular identity of fetal and subjects of similar weights (reviewed elsewhere ). One of the commonly studied adult preadipocytes has implications for research Subcutaneous and visceral adipose depots also human visceral adipose and physiology. First, expression of adiponectin in fetal diverge in their developmental formation and remodel- depots. The omental adipose precursors means that targeting adipocytes using the ling potential upon overnutrition. Monitoring de novo depot is inside the peritoneum, 76 starts near the stomach and commonly employed adiponectin promoter also targets adipocyte formation in mice revealed that subcutaneous spleen and extends deep into fetal adipocyte precursors, while this targeting is limi­ adipose commitment and differentiation are established the abdomen. ted to fully differentiated adipocytes in adults45. Careful by embryonic days 14–18, and in adults, this depot

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expands primarily through hypertrophy in response to cycling87,88, and fibroblast recruitment and extracellular overfeeding3. By contrast, the development of the vis- matrix deposition during wound healing89. ceral adipose depots occurs primarily postnatally, but Adipogenesis in the dermal adipose depot is most this tissue is capable of expanding through hyperpla- strongly established during wound healing. During sia as well as hypertrophy. Results utilizing alternative wound closure, adipocytes morphologically revert to a lineage-​tracing systems77 and stable isotope labelling78 fibroblast-like​ state through a process termed adipocyte– have led to similar conclusions. myofibroblast transition (AMT) to aid in wound closure. How can we reconcile the metabolically protective A subset of myofibroblasts at a cutaneous wound site expansion of subcutaneous fat in mice and humans with derive from an adiponectin-​positive lineage, implying the lack of observable preadipocyte hyperplasia within that they arise through an adipocyte de-​differentiation this depot in mouse models? We suggest two possible event65. Furthermore, there is increasing evidence that explanations. First, it is important to consider that the this transition is likely reversible, as complementary vast majority of studies of adipogenesis in rodents use line­age tracing has demonstrated that dermal adipocytes the C57/Bl6 mouse strain, partially because of the arise from the myofibroblast lineage following wound in­creased propensity of this strain to develop insulin resis­ closure64. While AMT may represent de-​differentiation tance to model metabolic decline associated with obesity. of adipocytes to a less lineage-​restricted myofibroblast, We cannot rule out that a genetically diminished capacity several studies also implicate the role of de novo adipo­ of subcutaneous hyperplastic expansion is both a con- genesis in wound healing. Genetically lipoatrophic tributor and a consequence of this genetic susceptibility mice (‘fatless’ mice, which are genetically incapable to insulin resistance, highlighting the need to expand of forming adipocytes) and mice treated with PPARγ the lineage-​tracking approaches to other mouse strains. antagonists to inhibit adipogenesis have profound Notably, however, when isolated and cultured in vitro, wound-​healing deficiencies89. This finding supports cells from the subcutaneous depot of C57/Bl6 mice the notion that adipocyte precursor proliferation may readily differentiate towards adipocytes79. In addition, be involved in the wound-​healing process. Clinically, certain metabolically healthy obese states (promoted preadipocytes (in clinical approaches referred to as by TZD treatment80,81 or transgenic overexpression of adipose stem cells) are gaining much attention for their adiponectin82) display an increase in subcutaneous adipo­ potential to improve healing in acute burns and chronic genesis. This observation suggests that subcutaneous non-​healing ulcers90,91, and they are the subject of a preadipocytes retain adipogenic capacity but are con- number of relevant clinical trials for and other non-​ strained in vivo under ‘normal’ experimental conditions healing skin conditions (NCT02590042, NCT03113747, of overnutrition. Stable isotope tracing of adipocyte turn- NCT02923219, NCT03427905 and NCT03264573). It over in mice also demonstrates that subcutaneous adi- is also of note that one of the most common sequelae pose tissue is capable of hyperplasia in juvenile mice but of clinical diabetes is impaired wound healing, and sev- not in adults78. This pattern of subcutaneous adipogenic eral studies note improved wound healing in patients potential followed by decline with ageing and obesity is with diabetes who were treated with adipose stem cell recapitulated in humans, where subcutaneous adipose therapy92,93. Classically, wound healing in people with depots of young, lean humans expand through adipogen- diabetes is attributed to increased inflammation or vas- esis during overfeeding83, but this capacity is diminished cular insufficiency, but these studies raise the possibility in individuals of advanced age and/or with obesity84. of increasing dermal adipocyte number and quality as a novel therapeutic avenue to improve the wound-​healing Adipogenesis outside of the main adipose depots. process in patients with diabetes. Apart from those in the main subcutaneous and visceral Adipocyte deposition in skeletal muscle is associated fat depots, adipogenic events have also been described in with insulin resistance, sarcopenia, muscular dystrophy other locations, including skin, skeletal muscle and bone and muscle injury94. In this case, the development of adipo­ marrow. Whereas adipogenesis appears to be beneficial cytes is associated with a decline in muscle function, in the skin, adipogenesis in the skeletal muscle and bone which is in contrast to the situation in adipose depots marrow is associated with pathology. of the skin. In early 2010, two independent studies95 Skin is the largest organ of the body and has complex identified that these infiltrating adipocytes in skeletal roles in immunity, thermoregulation and maintenance muscle arise from precursors different from muscle fibre of the mechanical integrity of the organism. Skin con- precursors, myoblasts. Uniquely, they further described tains many adipocytes that are anatomically localized the resident fibro-adipogenic​ progenitors (FAPs), which Panniculus carnosus directly below the dermis. These dermal adipocytes are expressed low levels of PPARγ at baseline and could be Striated muscle anatomically clearly distinct from those in the subcutaneous depot. prompted to undergo differentiation by niche factors located within or just beneath In rodents, the panniculus carnosus anatomically divides intrinsic to the injured muscle. New data suggest that the superficial fascia of the 85 dermis that controls local dermal from subcutaneous white adipose tissue . adipogenesis during muscle repair exacerbates muscle movement of the skin. It is Humans (and various other mammals such as pigs) lack injury. Reducing adipogenic differentiation of FAPs (by present in many lower a panniculus carnosus, but dermal adipocytes are ana- blocking Hedgehog signalling; see below for a discussion mammals but absent in tomically organized into ‘dermal cones’ superficial to the of signalling modulators of adipogenesis) enhances mus- humans. classical subcutaneous adipose tissue85. Adipocytes in cle regeneration during acute muscular injury and in a 96 Sarcopenia the skin are most highly appreciated for their mechanical model of muscular dystrophy . The age-related​ loss of muscle cushioning, but recent studies have revealed new roles The characteristically yellow appearance of bone mass. of dermal adipocytes in thermoregulation86, hair follicle marrow derives from a high quantity of adipocytes.

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Young mice and humans have a small quantity of mature (such as in subcutaneous adipose depots or under chow adipocytes within the bone marrow, but a great majority diet in mice)108. of the haematopoietic tissue of long is replaced These results raise the critical and thus far unaddres­ with adipocytes by early adulthood, which were long sed questions — what signals constrain or permit adipo­ thought of as simple filler for other bone marrow cells97. genesis in vivo, what are the cells of origin for these It is now appreciated that a variety of physiological fac- signals and how are they differentially regulated among tors, including obesity, anorexia, TZDs, glucocorticoids, adipose depots? These questions are especially difficult irradiation and ageing, can induce further differentiation to address because visceral and subcutaneous adipose of bone marrow adipocytes94. The apposition of adipo- depots have functionally different adipocytes and feature cytes and haematopoietic stem cells in bone marrow considerable differences of tissue organization, including underlies extensive crosstalk between the two tissues. different densities of innervation and vascularization. In Indeed, bone marrow adipocyte-​derived adipokines addition, recent single-cell​ sequencing data have demon- regulate haematopoietic regeneration98, as well as macro­ strated that the stromal populations of inguinal and phage99, B cell100 and development101. As in visceral adipose tissues contain different cell subpopu- other adipose depots, bone marrow adipocytes arise lations, some of which appear anti-adipogenic​ 109,110, indi- from a mesenchymal precursor shared with osteoblasts cating differences in stromal cell populations between and chondroblasts97. Uniquely, however, the preadipo­ the different depots. As the tools for studying these cytes of bone marrow are leptin receptor-​positive subpopulations of stromal cells improve, it seems likely (LepR+)102. Leptin has been shown to directly promote that they will provide insight into the niche-​specific adipogenesis of bone marrow mesenchymal precursors regulation of adipogenesis. and to inhibit osteogenic differentiation103. Impaired Single-cell​ sequencing reveals heterogeneity among leptin receptor signalling in bone marrow mesenchy- preadipocytes in various depots (Box 2), although it mal precursors reduces differentiation to adipocytes remains to be seen whether these preadipocytes rep- following fracture and improves bone regeneration in resent different stages of differentiation or whether mouse models103. Similarly, impairment of adipogenesis their molecular signatures are stable and characteristic (in lipoatrophic mice or induced by treatment with a of certain depots109. Some data also suggest an intrin- PPARγ inhibitor) after irradiation improves bone mar- sic limitation to the plasticity of adipose precursors. row engraftment and supports haematopoietic recov- For example, brown adipocytes but not white or beige ery104. Interestingly, leptin does not appear to be required adipocytes derive from a myogenic factor 5-positive for adipogenesis following bone marrow irradiation104, (MYF5+) lineage (Box 1), and suppression of brown implying that different signals may influence adipogen- adipogenesis through deletion of the thermogenic esis after different injuries. More recently, a separate transcription co-​regulator PRDM16 promotes acqui- study identified the adipogenic fraction of mesenchy- sition of muscle morphology but not white adipocyte mal precursors that proliferate after bone fracture as morphology111. By contrast, deletion of PRDM16 dur- ZFP423+ cells and demonstrated that increased adipo- ing beige adipogenesis promotes white adipocyte dif- genesis is sufficient to inhibit fracture healing105. Thus, ferentiation112. This suggests the existence of lineage in both muscle and bone marrow, selective repression plasticity between white and beige adipocytes that is of local adipogenesis following a traumatic event is not conserved in brown adipocytes. Notably, the point a new and promising therapeutic strategy to improve of divergence of white and brown adipocyte lineages is tissue regeneration. controversial, because some studies find some contri- bution of MYF5+ progenitors to the white adipose line- Importance of the niche in adipogenesis. Given the age113, suggesting greater potential for lineage plasticity heterogeneity­ and differences in the function of adipo­ in favourable conditions. genesis in different niches, a natural question arises: what defines these differences? Signalling modulators of adipogenesis A number of observations indicate a high degree Many important signalling hormones and ligands mod- of plasticity of preadipocytes and implicate local niche ulate the process of preadipocyte differentiation into a factors in regulating adipogenesis. For example, donor mature adipocyte (Fig. 4). preadipocytes isolated from subcutaneous or visceral depots behave in a manner consistent with the site Insulin signalling. Insulin is an anabolic pancreatic pep- of injection in the recipient rather than their depot of tide hormone secreted in response to increased plasma origin during high-​fat feeding (undergoing differen- glucose to promote plasma glucose uptake into periph- tiation in the visceral but not subcutaneous depot)106. eral tissues, such as skeletal muscle and adipocytes, for This plasticity of preadipocytes is further implied by utilization or storage. Insulin signals through high-​ recent observations that PDGFRα+ cells are able to form affinity binding to the insulin receptor in specialized either beige adipocytes in response to β3-adrenergic tissues or with lower-affinity​ binding through the related stimulation (Box 1) or phenotypically white adipocytes insulin-like​ growth factor 1 (IGF1) receptor found on all during high-​fat feeding107. Perhaps most striking is cells. Intracellularly, the insulin-​signalling cascade then the new report that PPARγ expression, though suffi- activates insulin receptor substrates (primarily IRS1) cient to drive adipogenesis in cultured fibroblasts51, is and then PI3K and AKT1 or AKT2 kinase. This cascade insufficient to stimulate adipogenesis in vivo in con- leads to the activation of CREB, mTOR and the family texts when adipogenesis is physiologically limited of forkhead proteins (FOXOs)114.

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Signalling Systemic expressed at much higher levels in preadipocytes than insulin receptors are, higher levels of insulin in vivo • Basal ROS levels: • Inhibition of adipocyte preadipocyte differentiation would be required to activate intracellular insulin sig- expansion • Insulin resistance nalling in undifferentiated preadipocytes than needed Insulin • High ROS levels: for mature adipocytes128. However, many unaddressed adipocyte TNF dysfunction TGFβ questions relating to the role of insulin in adipogenesis remain. Importantly, understanding of how preadipo- AKT IL-1β SHH ROS cytes discriminate between normal postprandial insulin mTOR IL-6 elevations and insulin elevations associated with over­ NO nutrition is elusive. Along similar lines, it is unclear how PPAR γ MCP1 the response to insulin is regulated in preadipocytes, and BMP SMAD4 Chronic inflammation signals that facilitate or oppose insulin signalling need C/EBPα to be refined. β-Catenin Circadian regulation of PPARγ expression signalling. Glucocorticoids are steroid WNT hormones that signal through the nearly ubiquitously GCR Circadian GC expressed glucocorticoid receptor to suppress inflam- rhythms mation and promote mobilization of nutrients from GC metabolic tissues. In healthy individuals, plasma gluco- • Promotes brown/beige corticoids peak shortly after waking from sleep and fall adipogenesis throughout the rest of the day129, whereas in individuals • Increases energy expenditure with obesity, elevated levels of plasma glucocorticoids persist130,131. Glucocorticoids are one of the three crit- Cold exposure ical components of in vitro adipocyte differentiation Fig. 4 | regulators of adipogenesis. Many extracellular signals are reported to influence medium and are thus considered crucial pro-adipogenic​ whether a preadipocyte differentiates. These can broadly be divided into signalling signals132,133. In preadipocytes, glucocorticoids facilitate molecules and systemic physiological factors. Specific signalling ligands, including growth arrest134 and are required for terminal differen- insulin, glucocorticoids and bone morphogenetic proteins (BMPs), directly activate tiation. They also upregulate several transcription fac- peroxisome proliferator-activated​ receptor-γ​ (PPARγ) and/or CCAAT/enhancer-binding​ tors required for differentiation, including C/EBPs135,136. protein-​α (C/EBPα) to facilitate preadipocyte differentiation. Other signalling inputs, Moreover, glucocorticoid signalling sensitizes preadipo- such as those of the WNT and Hedgehog families of ligands, suppress adipogenesis cytes to insulin signalling137, enhancing the adipogenic through direct repression of the PPAR –C/EBP complex or through suppression of γ α actions of the insulin pathway. other pro-adipogenic​ signalling cascades, such as inhibition of insulin signalling by Hedgehog. Separately , systemic physiological states, such as those associated with oxidative stress and the production of reactive oxygen species (ROS), inflammation, BMP signalling. BMPs are a class of conserved signalling circadian rhythms and cold temperatures, have complex and context-dependent​ effects molecules in the transforming growth factor-​β (TGFβ) on adipogenesis and metabolic health. IL , interleukin; TGFβ, transforming growth family of proteins, originally identified for their ability to factor-β; TNF, tumour necrosis factor. induce bone and formation but now known to act as growth factors throughout the body. BMP sig- nalling through BMP receptors results in the intracellular Insulin is an essential component of in vitro adipo­ phosphorylation and activation of SMAD proteins. As cyte differentiation medium, and highly adipogenic previously discussed, BMP signalling is required for cells will differentiate spontaneously in vitro in the adipogenesis in vitro41–43, and active SMAD4 results in presence of insulin without other hormonal cocktails. transcription of PPARγ and facilitation of adipogen­ Disruption of the components of the insulin signalling esis42. Different members of the BMP family have been cascade, including the insulin receptor115,116, IRS family shown to selectively promote brown138 or white139 adipo- members117,118, PI3K119,120, AKT1 or AKT2 (refs121–123), cyte differentiation. Consistent with a role of BMP4 as a mTOR124,125 and the FOXO family126, leads to failure of pro-adipogenic​ factor for white adipose tissue, individu- adipogenesis in vitro. In vivo, elimination of the insulin als identified as obese but without diabetes have elevated receptor from late-stage​ differentiating adipocytes using circulating BMP4 (ref.140), suggesting that the elevation an AP2-promoter-​driven Cre recombinase curiously of BMP signalling allows for adequate adipogenesis to does not lead to a failure of adipogenesis, suggesting that avoid metabolic decline in obesity. the IGF1 receptor is capable of compensating for the loss of the insulin receptor in vivo (at least during the late WNT signalling. WNTs are a highly conserved family stages) provided the downstream signalling is intact127. of autocrine and paracrine ligands known for their roles Considering the physiological role of insulin in in embryonic development and carcinogenesis. Despite glucose metabolism, an interesting implication is that their generally pro-​growth roles in these processes, insulin serves as a permissive differentiation signal to WNTs are perhaps the most established suppressors of preadipocytes. Persistent elevation of plasma insulin adipogenesis141. Canonical WNT signalling results in the is a hallmark of overnutrition, and a logical adaptive stabilization of β-​catenin. In preadipocytes, this stabili- response to this overnutrition would be to increase the zation results in a failure of PPARγ and C/EBPα induc- adipocyte number to create additional safe storage sites tion and, in some cases, a shift towards an osteoblastic for macronutrients. Considering that IGF1 receptors are or immune cell phenotype141–143. In vivo, hypoxia144,145

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and the vascular endothelium146 were shown to increase do not necessarily need to adopt a macrophage pheno- WNT levels, providing an interesting means of limiting type to be inflammatory159. A subset of fibroblast-​like adipogenesis in regions where the vasculature is unable PDGFRα+ precursors marked by high expression of the to support additional adipocytes. cell surface marker CD9, which are increased in indi- viduals with diabetes, were found to contribute to tis- Hedgehog signalling. The Hedgehog family of ligands is sue inflammation and fibrosis, whereas precursors with conserved from flies to humans and is critically involved low CD9 expression, which are capable of adipogenesis, in body patterning during embryogenesis. Hedgehog were depleted in individuals with diabetes159. Such fibro-​ signalling decreases adipogenesis in rodent fibroblasts inflammatory progenitors (FIPs) were independently by interfering with BMP signalling147 and insulin sig- described in another study as a distinct cell population nalling148. Interestingly, Hedgehog signalling appears to of PDGFRβ+ perivascular cells within visceral adipose redirect adipose precursors towards an osteogenic fate depots of mice. These FIPs were shown to express higher in vivo149. It also restricts adipogenesis in the muscle levels of inflammatory cytokines than regular adipocyte after an injury to promote healing96. precursors and responded more strongly to inflam- matory stimuli. Most surprisingly, FIPs could directly Systemic regulation of adipogenesis repress the differentiation of adipogenic precursors. This Several key physiological states and cues can influ- work raises the interesting possibility of heterogeneity ence adipogenesis (Fig. 4). These are more integrative within the adipogenic lineage in vivo and provides new than simple signalling inputs and could modulate insights into the mechanisms underlying the detrimental adipogenesis more globally. role of inflammation on adipogenesis160. Importantly, we draw a distinction between acute Inflammation. Chronic, low-grade​ inflammation is one and chronic inflammation. Chronic inflammation con- of the hallmarks of diet-​induced metabolic disease. The tributes to the pathophysiology of most adipose tissue low-level​ inflammation during obesity is clearly different dysfunction, including the impairment of adipogenesis from the inflammation evoked during an acute infec- that contributes to insulin resistance and metabolic dis- tion but involves many of the same players, including ease. Notably, however, transient acute inflammation of . adipose depots seems to have a beneficial role in adipo- Early studies utilizing in vitro differentiation of 3T3- genesis and metabolic homeostasis. For example, TGFβ L1 fibroblasts demonstrated that the addition of pro-​ signalling, when induced acutely, was shown to promote inflammatory factors, such as TNF and IL-6 cytokines, beige adipogenesis in white adipose depots161–163, which to the culture medium impairs adipocyte differenti- is metabolically beneficial. By contrast, impairing adi- ation150. Macrophages are the main inflammatory cell pose tissue inflammation during high-fat​ feeding is asso- type infiltrating adipose tissue and contribute consid- ciated with decreased adipogenesis in vivo (although no erably to the local levels of these pro-​inflammatory defects are observed in vitro)164. These results suggest agents during high-​fat feeding151. Interestingly, it has that transient, localized inflammation may be a potent been demonstrated that a strong positive correlation stimulant of preadipocyte differentiation. exists between adipocyte size and the extent of macro­ In summary, we find that transient inflammation phage infiltration in white adipose tissue151,152. This is likely an important part of the tissue remodelling finding raises the question — does in vivo inflammation process required for adipogenesis. However, sustained suppress adipogenesis or is inflammation a secondary chronic inflammation likely derives from insufficient consequence of unhealthy adipose expansion? adipogenesis within adipose tissue and forms a vicious The most established example for an anti-adipogenic​ cycle where inflammation further inhibits adipogenic inflammatory molecule is TGFβ. TGFβ is secreted precursor differentiation. from hypertrophic, dysfunctional adipocytes and from immune cells recruited to the adipose depot during Circadian rhythms. Peripheral and central circadian obesity in mice and humans153,154. Since the 1980s, ele- oscillators are important to coordinate day and night vated TGFβ has been known to inhibit adipocyte dif- cycles, behaviour (including feeding) and the overall ferentiation in vitro155, and mice overexpressing TGFβ physiological state. Disruption of normal circadian pat- have a lipodystrophic phenotype with severely impaired terns in humans (for example as a result of shift work) adipocyte differentiation156. Molecularly, TGFβ signals increases the risk of obesity, including abdominal obe- through SMAD3 and directly inhibits PPARγ–C/EBPα sity associated with the metabolic syndrome165. Emerging complex formation and, in consequence, adipogen­ evidence from mouse models suggests that the increased esis. Other inflammatory factors, such as TNF, are also adiposity in individuals with disrupted circadian rhyth- Metabolic syndrome known to directly impair adipogenesis157 but also pro- micity is regulated at the level of the preadipocyte. The A combination of five metabolic risk factors: high mote the elevation of TGFβ as part of their downstream expression of the master adipogenic factor, PPARγ, is blood pressure, elevated signalling, creating a synergistic effect. cyclic in white adipose tissue and liver but not in other fasting blood glucose, hypertri- Preadipocytes can adopt a macrophage-like​ inflam- peripheral metabolic tissues such as brown adipose glyceridaemia, decreased matory phenotype with increased expression of pro-​ tissue or skeletal muscle166. Preadipocytes also show serum high-density​ lipoprotein and increased abdominal inflammatory cytokines and decreased adipogenic specific oscillations in the expression of core circadian 167 adiposity, which collectively capacity in response to inflammatory stimuli, which clock components, including PER2 and PER3 (ref. ). increase the risk of heart can eventually lead to adipose tissue fibrosis143,158. Molecularly, both PER2 and PER3 restrain adipogenesis disease, diabetes and stroke. A recent study suggested that adipogenic precursors — PER2 through direct repression of PPARγ168 and PER3

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through the repression of KLF15, which was shown to ROS by interfering with the mitochondrial respiratory promote adipogenesis of 3T3-L1 cells167, or potentially chain decreases preadipocyte differentiation in vitro, also through direct repression of PPARγ169. Additionally, which was reversible with antioxidant treatment174. the circadian oscillator BMAL1 and its downstream tar- However, compelling and seemingly contradictory get, nocturnin, both oscillate in an antiphasic manner data demonstrate that ROS may facilitate adipocyte to the PER paralogues and promote adipogenesis170 by differentiation. The addition of 100 µM hydrogen per- enhancing PPARγ nuclear localization171. oxide to a standard hormonal differentiation cocktail Crucially, these studies implicate circadian cycling markedly improved adipogenic differentiation in the as a regulator of adipogenesis. However, a true under- 3T3-L1 preadipocyte cell line. Interestingly, this effect standing of the physiological impact of these factors was stronger in the absence of insulin175. Additional is still missing. Most mouse models used to study cir- work showed that in human mesenchymal stem cells cadian core components use constitutively null alleles, differentiating into adipocytes, generation of ROS is pro- which are often deleted in multiple tissues other than moted by mTORC1 through activation of mitochondrial preadipocytes (that is systemic deletions, adipocyte electron transport complex III. In addition, antioxidants deletions and others). Such manipulations pose a risk of targeted to the mitochondria are capable of substantially alterations in adipose tissue development, which would reducing adipocyte differentiation in vitro176. Since the confound interpretation of adipogenic phenotypes in publication of these initial results, several independent adults. Nonetheless, we can envision a scenario where groups have further confirmed that decreasing cellular the correct temporal combination of a circadian signal ROS or providing an extracellular antioxidant impairs and a systemic or niche differentiation signal must align adipocyte differentiation177–179. to induce an adipogenic event. A proof of concept for At a molecular level, there appears to be a high level this systemic cue–circadian coordination of adipogen­ of crosstalk between insulin signalling, ROS and adipo­ esis is observed with the temporal requirements for glu- genesis. Insulin signalling generates ROS through acti- cocorticoid signalling in differentiating preadipocytes. vation of the NADPH oxidase NOX4, which in turn In healthy animals, plasma glucocorticoids oscillate, inhibits PTP1B, an intracellular protein phosphatase with elevations during the active (awake) period. These and inhibitor of the insulin signalling pathway180,181. oscillations flatten in obese animals, leading to persis- This property of ROS is associated with the enhance- tently high levels of circulating glucorticoids. Similar to ment of insulin signalling and increased adipogenesis. the persistent glucocorticoid signal in individuals with However, sustained high levels of ROS produced from obesity that facilitates adipogenesis, it is standard to NADPH oxidases during unhealthy obesity182 inhibit induce adipogenic differentiation in vitro with approxi­ insulin signalling and thus inhibit adipogenesis183,184. mately 24 hours of treatment with the corticosteroid dexa­ How do we reconcile these seemingly paradoxical methasone, which is an agonist of the glucocorticoid results? One interesting possibility is that there is a cer- receptor. However, if this same total dose of dexametha­ tain basal level of ROS required for preadipocyte dif- sone is fractionated into 12-hour-​on and 12-hour-​off ferentiation. It is possible that a mild exogenous ROS increments, then preadipocytes fail to differentiate172. signal sent from adipocytes to precursors serves as a Importantly, if the temporal exposure to dexametha- developmental cue, promoting adipogenesis, whereas sone is lengthened to 18 hours or longer, preadipocytes high levels of ROS in the tissue cause cell dysfunction undergo adipogenic differentiation, suggesting the and damage. Understanding the physiological sources length of exposure to glucocorticoid signalling regulates of ROS during adipogenesis and the thresholds at which differentiation independently of the total dose172. pro-​adipogenic and anti-​adipogenic responses occur is This suggests that the controlled elevation of hor- critical to reconciling this seeming paradox. mone levels allows for temporal coordination of PPARγ activity with the preadipocyte clock programme. Again, Adipogenesis in health and disease these results are limited to in vitro studies but support Lack of developmental adipogenesis manifests as lipo- a model for systemic and circadian coordination of dystrophies (characterized by a complete or partial adipogenesis. lack of adipose tissue), which has considerable detri­ mental effects on systemic glucose and lipid meta­ Reactive oxygen species. Reactive oxygen species (ROS) bolism. Adipose tissue mass also declines with ageing, are molecules including free oxygen and superoxide with which could contribute to metabolic decline in old age. an unpaired electron. Normal mitochondrial oxidative Finally, because adipose tissues support various aspects metabolism generates a low amount of ROS, usually of metabolism, insufficient adipogenesis during obesity quenched by intracellular antioxidant enzymes. However, exacerbates the metabolic syndrome. if the amount of ROS generated exceeds the buffering capacity of the cell, these electrophilic ROS damage DNA Perturbation of developmental adipogenesis in and RNA and oxidize lipids and amino acids in a pro- lipodystrophies. Lipodystrophies are a class of meta- cess known as oxidative damage173. In liver, adipocytes bolic diseases caused by a decreased amount (or near and other metabolically active tissues, oxidative damage, complete absence) of adipose tissue. Individuals with especially to mitochondrial electron-​transport-chain lipodystrophies have very limited fat tissues but have components, leads to inefficient ATP production and profound insulin resistance and dyslipidaemia, because metabolic dysfunction173. In preadipocytes, early studies the lipid ordinarily stored in adipose tissue prematurely demonstrate that increased generation of intracellular accumulates in secondary peripheral tissues.

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Caveolae Congenital generalized lipodystrophy (CGL) is the much more varied in FPLD than in CGL, and leptin Small, flask-​like invaginations most severe form of lipodystrophy and presents as supplementation remains an efficacious treatment for of plasma membrane that are almost no adipose tissue in all locations from infancy. patients, especially in those with marked hypoleptinae- abundant in many mammalian Several mutations have been identified to cause CGL, mia. However, unlike CGL, there is some evidence that cell types including adipocytes. They have been implicated in including mutations in the lysophosphatidic acid acyl- TZDs may be efficacious at reversing the adipose tissue 185 various processes, including transferase, AGPAT2 (ref. ), the lipid droplet associated loss associated with FPLD and consequently alleviating endocytosis, signalling, lipid protein BSCL2 (ref.186), the plasma membrane scaffold- metabolic dysfunction204–206. regulation and ing proteins associated with caveolae CAV1 (ref.187) mechanosensing. and PTRF188. AGPAT2 and BSCL2 are crucial for lipid Blunted adipogenesis during ageing. Ageing is gener- biosynthesis and storage, a process required in adipo- ally associated with organ and tissue functional decline, cyte formation189,190. Specifically, AGPAT2-deficient and adipose tissue is no exception. Loss of subcutaneous preadipocytes fail to induce phospholipases required adipose tissue mass along with the progressive ectopic for terminal adipocyte differentiation, whereas BSCL2- lipid deposition in muscle and liver is a hallmark of age-​ deficient preadipocytes fail to sustain an upregulation related decline and frailty (reviewed elsewhere207). Ageing of PPARγ, C/EBPα and other terminal adipocyte dif- is also associated with the decline in adipogenic potential, ferentiation drivers in late-​stage adipocyte differentia- which is caused by the acquisition of a senescent pheno­ tion191,192. Caveolae are induced nearly tenfold during type by preadipocytes, which restricts preadipocyte adipogenesis193, and mice with deficiencies of CAV1 proliferation and differentiation capacity and contribu­ partially mimic the human lipodystrophic phenotype tes to adipose tissue deterioration during ageing78,208,209. with severe adipose tissue dysfunction194. Although In addition, these senescent preadipocytes actively not tested directly, CAV1 may enhance adipogenesis decrease adipose tissue insulin sensitivity through secre- through crosstalk with the insulin signalling pathway, tion of pro-inflammatory cytokines, such as TNF210 and as it has been shown to enhance intracellular insulin IL-6 (ref.211), both of which also inhibit adipogenesis­ 150–152. signalling195. Similarly, PTRF is required for functional Senescent preadipocytes were also shown to secrete caveolae formation, and lack of PTRF prevents proper activin A, a protein belonging to the TGFβ super­ formation and release of caveolae from cells196. CAV1- family, which similar to TGFβ, inhibits adipogenesis210. containing extracellular vesicles are essential for cross- Pharmacological depletion of senescent cells is sufficient talk between cells in adipose tissue in vivo197, and loss of to maintain preadipocyte adipogenic capacity and pre- this communication may reduce crucial cell–cell adipo- serves adipose tissue function in old age212,213. Targeting genic signalling. Interestingly, extracellular vesicles are and reversing cellular ageing of preadipocytes are inter- gaining attention for their potential therapeutic use198, esting therapeutic concepts to maintain adipose tissue and restoration of extracellular-​vesicle-mediated com- homeostasis and metabolic health in aged individuals. munication in adipose tissue is an exciting new target for Furthermore, hyperplasia of adipose tissue can have some of these most severe lipodystrophies. indirect benefits on lifespan. Decreased insulin levels and Interestingly, both humans199 and mice200 with com- insulin signalling are among the strongest factors asso- plete loss of adipose tissue show robust metabolic ciated with extended lifespan214. As illustrated in this improvement with leptin administration. However, lep- Review, adipogenesis during overnutrition preserves tin does not restore fat mass; rather, it only attenuates metabolic health, leading to systemically decreased insu- insulin resistance and dyslipidaemia. Using the inducible lin levels, and dampens insulin signalling, thus potentially fatless mouse (in which the loss of adipose tissue can be indirectly contributing to extended lifespan. Additionally, induced specifically in the adult), it was established that several adipokines including adiponectin30 and FGF21 the loss of adipose tissue together with the loss of leptin (ref.215) correlate with extended lifespan. FGF21 can presents with a more severe metabolic dysfunction than directly activate PPARγ to promote adipogenesis216, in the absence of leptin alone201. This finding suggests that and there is evidence that smaller cells formed through the adipose tissue has profound regulatory inputs into sys- adipogenesis express higher levels of adipo­nectin17. temic metabolism, beyond secretion of adipokines, and Taken together, these studies suggest the presence of a that restoration of adipose mass, in addition to the mere healthy, self-​reinforcing cycle whereby healthy hyper- restoration of adipokine levels, is a beneficial therapeutic plastic expansion of adipose tissue leads to a longevity-​ avenue for lipodystrophy patients in the future. inducing adipokine profile, which in turn maintains Familial partial lipodystrophy (FPLD) has a similar systemic insulin sensitivity and metabolic health and presentation to CGL, except the loss of adipose tissue could feed back to further promote adipogenesis. is mainly from the subcutaneous depot. Mutations in lamin A and PPARγ are most common, although muta- Promoting adipogenesis for metabolic health. Many tions in lipid droplet biogenesis and metabolism factors years of research using humans and animal models (perilipin 1, CIDEC and lipase E) and in the adenosine have established a strong correlation of adipogenesis and 2 A receptor, which is implicated in thermogenesis in preserved metabolic health. brown and beige adipocytes, have been reported202. More recently, genome-wide​ association studies have Deficiencies in adipogenesis resulting from mutations identified several loci involved in adipocyte differentia- in PPARγ are not surprising, but it is interesting to note tion as susceptibility loci for the development of insulin that mutations in lamin A, which is a ubiquitous nuclear resistance and ectopic fat deposition217,218. An interest- envelope protein, also cause primary defects in adipocyte ing question then arises: can simply increasing adipose differentiation203. The clinical spectrum of symptoms is tissue mass enhance metabolic health?

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Emerging work in mouse models suggests that Right now, the question of whether adipogenesis con- improved metabolic health in obese animals can fers a benefit to metabolically healthy individuals needs be induced upon further, healthy expansion of fat further exploration. It is challenging to manipulate adipo- mass82,219–221. In these mice, adipogenesis allows adi- genesis in animal models without also influencing con- pose tissue to expand while limiting hypoxia, chronic founding factors, such as total adiposity, especially in light inflammation and fibrosis82,219. In the absence of these of the fact that physiological rates of adipogenesis in pathologies, adipose tissue metabolic function is fully lean animals are very low. Additionally, multiple physio­ preserved. These findings raise the interesting possibility logical brakes on adipogenesis exist in vivo (for exam- that obesity-​associated metabolic decline is not due to ple, overexpression of PPARγ in PDGFRβ+ perivascular adiposity, per se, but rather is a result of an insufficient cells does not drive differentiation under standard chow capacity of adipose tissue to further expand. This leads feeding conditions108), and multiple cues are required to adipose tissue dysfunction and persistently elevated to induce adipocyte hyperplasia. It is also important to levels of plasma glucose and lipids, which accumulate in note that, before the adoption of in vivo lineage-​tracing other tissues and contribute to insulin resistance. models, adipogenesis was quantified in vivo by examin- Several transgenic mouse models of metabolically ing adipose cellularity, which would not detect changes healthy obesity from our group and others demonstrate in adipocyte turnover (balanced adipogenesis and apop- considerable adipose tissue expansion in compari- tosis of adipocytes), which may confer an independent son with that of the wild type but with the presence of metabolic benefit by maintaining a constant adipose tis- smaller adipocytes219,222,223. Despite their increased body sue mass. The difficulty of generating a lean mouse with mass compared with controls, these healthy obese mice increased adipogenesis is an endeavour, but such models all display extraordinarily preserved systemic meta- are required to determine whether adipogenesis, per se, bolic health. It is important to note, however, that these confers metabolic benefits. mouse models certainly represent genetic manipulations Correlative studies in the clinic provide clues that (overexpression of factors promoting adipogenesis) and adipogenesis in lean individuals may indeed be benefi- almost certainly also have developmental differences cial. For example, humans predisposed to hypertrophic in adipose tissue formation. However, these mod- obesity have reduced insulin sensitivity, even in the els demonstrate that excess calories stored in healthy, lean state227,228, and this is a common feature seen for hyperplastic adipose tissue do not contribute to meta­ individuals without obesity but with clinical insulin bolic decline. Consistent with the notion of healthy resistance229. Furthermore, follow-​up studies of babies hyperplastic expansion in obesity demonstrated in these born during food shortages in Europe indicated that a mouse models, treatment with TZDs to activate PPARγ decreased birthweight predisposes the child to obesity has been reported to increase adipose tissue mass, and insulin resistance later in life230–232. While these early decrease adipo­cyte size and improve systemic meta­ studies predominantly used birthweight as a surrogate bolism in rodents and humans224,225. The expansion of for adiposity, several hints suggest that adipogenesis is the subcutaneous fat tissue is an integral component specifically impaired. Maternal food restriction during of these improvements. the second trimester of pregnancy, when adipose tissue More recently, studies in several new mouse mod- is actively being formed, results in severe metabolic els have highlighted the important role for hyperplas- impairments later in life, while restriction in the third tic expansion of the visceral depot in metabolic health trimester, after the tissue is formed, does not produce maintenance. Tenomodulin is a transmembrane protein such lifelong metabolic consequences232. preferentially enriched in the adipocytes of obese indi- There is also evidence that too much adiposity at viduals226. Notably, tenomodulin has been recently shown birth also predisposes individuals to metabolic disease. as a factor required for adipogenesis, whereby constitu- Cross-​sectional studies comparing babies of European tive overexpression of tenomodulin in adipocytes leads descent with Indian babies demonstrate that Indian to visceral adipose tissue expansion through adipo- babies of the same birthweight have relatively higher adi- genesis220. Despite the fact that high visceral adi­posity posity than their European-​descent counterparts233,234. usually correlates with metabolic decline, the adipo­ This increased abdominal adiposity at birth carried an genic nature of the expansion in this particular mouse increased risk of obesity and diabetes in adulthood, sug- model allows for improved metabolic health compared gesting that fetal expansion of abdominal adipose tissue with control animals. In another study, inhibiting adult may not be desirable. adipogenesis of PDGFRβ+ perivascular cells through There is clearly a high degree of crosstalk between deletion of PPARγ led to a pathological, hypertrophic maternal nutrition state, fetal adipose tissue develop- white adipose tissue expansion upon high-fat-diet​ feed- ment and lifelong risk of developing metabolic disease. ing108. Conversely, stimulating adult visceral adipogen- Greater clarity about which factors influence adipogen- esis during overnutrition by overexpression of PPARγ esis during which stages of fetal development has enor- had beneficial effects, including reduced levels of local mous public health potential and is a very promising inflammation, preserved serum adiponectin levels and area for future study. improved metabolic health, without impacting body weight or adiposity108. These data provide proof of con- Conclusions and perspective cept that expanding the number of visceral adipocytes We are beginning to understand the complex web of mole­ can occur without increasing overall adiposity and body cular components and cues governing adipocyte differ- weight, per se, even under increased caloric intake. entiation. Several key themes emerge. First, adipogenesis

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is a physiological process that enhances the ability of In addition, adipogenic precursors themselves — by the tissue to safely sequester lipids and prevent lipo- modulating tissue inflammation — can impact adipo- toxicity in peripheral organs. This is true during fetal genesis. Finally, it is now evident that adipose depot development, where defective adipogenesis leads to health is more important than its size alone and that lipodystrophies, as well as in adulthood, where pre- increased adipogenesis during weight gain can offset the served metabolic health during obesity correlates with negative metabolic consequences of high fat exposure. and is facilitated through adipogenesis. Second, pread- Overall, adipogenesis now emerges as a viable ther- ipocytes show remarkable heterogeneity and lineage apeutic target. However, PPARγ agonists (TZDs) used plasticity and likely are perivascular. White adipose tis- as pro-​adipogenic compounds were met with mixed sue contains a set of preadipocytes capable of produc- enthusiasm and caused controversy upon their initial ing both white and beige adipocytes in vivo. Adipose introduction to the market owing to the associated precursors are also found outside of adipose depots weight gain frequently observed and certain cardiac side and have important roles (beneficial or detrimental) effects (which later proved to be of no major concern). in skin, muscle and bone homeostasis. It has also been We believe that an increased understanding of adipo- recently appreciated that adipocytes may return to a genesis holds great promise as a new avenue to treat fibroblast (‘preadipocyte-​like’) state, suggesting that various diseases, including metabolic disorders, lipo­ adipocyte differentiation may not be terminal as previ- dystrophies and healing and regeneration of tissues, such ously thought. Third, local signalling and systemic cues, as skin, muscle and bone marrow, which should be made such as inflammation, ROS and other factors, influence possible with locally acting modulators of adipogenesis. adipogenesis. There is also evidence that a niche can Published online xx xx xxxx modulate preadipocyte fate, fine tuning adipogenesis.

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