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

Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of β-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.

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

Adipose tissue, once thought to be an inert receptacle for energy storage, is now recognized as a complex tissue with multiple resident cell populations that actively collaborate in response to diverse local and systemic metabolic, thermal, and inflammatory signals. A key participant in adipose tissue homeostasis that has only recently captured broad scientific attention is the lymphatic vasculature. The lymphatic system's role in lipid trafficking and mediating inflammation makes it a natural partner in regulating adipose tissue, and evidence supporting a bidirectional relationship between lymphatics and adipose tissue has accumulated in recent years. Obesity is now understood to impair lymphatic function, whereas altered lymphatic function results in aberrant adipose tissue deposition, though the molecular mechanisms governing these phenomena have yet to be fully elucidated. We will review our current understanding of the relationship between adipose tissue and the lymphatic system here, focusing on known mechanisms of lymphatic-adipose crosstalk.

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

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

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

The lymphatic vasculature plays important roles in the physiology of the organs in which it resides, though a clear mechanistic understanding of how this crosstalk is mediated is lacking. Here, we performed single-cell transcriptional profiling of human and mouse adipose tissue and found that lymphatic endothelial cells highly express neurotensin (NTS/Nts). Nts expression is reduced by cold and norepinephrine in an α-adrenergic-dependent manner, suggesting a role in adipose thermogenesis. Indeed, NTS treatment of brown adipose tissue explants reduced expression of thermogenic genes. Furthermore, adenoviral-mediated overexpression and knockdown or knockout of NTS in vivo reduced and enhanced cold tolerance, respectively, an effect that is mediated by NTSR2 and ERK signaling. Inhibition of NTSR2 promoted energy expenditure and improved metabolic function in obese mice. These data establish a link between adipose tissue lymphatics and adipocytes with potential therapeutic implications.

Adipose thermogenesis is repressed in obesity, reducing the homeostatic capacity to compensate for chronic overnutrition. Inflammation inhibits adipose thermogenesis, but little is known about how this occurs. Here we showed that the innate immune transcription factor IRF3 is a strong repressor of thermogenic gene expression and oxygen consumption in adipocytes. IRF3 achieved this by driving expression of the ubiquitin-like modifier ISG15, which became covalently attached to glycolytic enzymes, thus reducing their function and decreasing lactate production. Lactate repletion was able to restore thermogenic gene expression, even when the IRF3/ISG15 axis was activated. Mice lacking ISG15 phenocopied mice lacking IRF3 in adipocytes, as both had elevated energy expenditure and were resistant to diet-induced obesity. These studies provide a deep mechanistic understanding of how the chronic inflammatory milieu of adipose tissue in obesity prevents thermogenic compensation for overnutrition.

OBJECTIVE: Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity.

METHODS: We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes.

RESULTS: Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFβ1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGFβ1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFβ1.

CONCLUSIONS: Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFβ-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.

The chemokine CCL2 (also known as MCP-1) is a key regulator of monocyte infiltration into adipose tissue, which plays a central role in the pathophysiology of obesity-associated inflammation and insulin resistance. It remains unclear how CCL2 production is upregulated in obese humans and rodents. Because elevated levels of the free fatty acid (FFA) palmitate and TNF-α have been reported in obesity, we studied whether these agents interact to trigger CCL2 production. Our data show that treatment of THP-1 and primary human monocytic cells with palmitate and TNF-α led to a marked increase in CCL2 production compared with either treatment alone. Mechanistically, we found that cooperative production of CCL2 by palmitate and TNF-α did not require MyD88, but it was attenuated by blocking TLR4 or TRIF. IRF3-deficient cells did not show synergistic CCL2 production in response to palmitate/TNF-α. Moreover, IRF3 activation by polyinosinic-polycytidylic acid augmented TNF-α-induced CCL2 secretion. Interestingly, elevated NF-κB/AP-1 activity resulting from palmitate/TNF-α costimulation was attenuated by TRIF/IRF3 inhibition. Diet-induced C57BL/6 obese mice with high FFAs levels showed a strong correlation between TNF-α and CCL2 in plasma and adipose tissue and, as expected, also showed increased adipose tissue macrophage accumulation compared with lean mice. Similar results were observed in the adipose tissue samples from obese humans. Overall, our findings support a model in which elevated FFAs in obesity create a milieu for TNF-α to trigger CCL2 production via the TLR4/TRIF/IRF3 signaling cascade, representing a potential contribution of FFAs to metabolic inflammation.