Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. METHODS: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. RESULTS: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. CONCLUSIONS: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

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

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

Aging impairs cold-induced beige adipogenesis and adipocyte metabolic reprogramming.

The energy-burning capability of beige adipose tissue is a potential therapeutic tool for reducing obesity and metabolic disease, but this capacity is decreased by aging. Here, we evaluate the impact of aging on the profile and activity of adipocyte stem and progenitor cells (ASPCs) and adipocytes during the beiging process. We found that aging increases the expression of Cd9 and other fibro-inflammatory genes in fibroblastic ASPCs and blocks their differentiation into beige adipocytes. Fibroblastic ASPC populations from young and aged mice were equally competent for beige differentiation in vitro, suggesting that environmental factors suppress adipogenesis in vivo. Examination of adipocytes by single nucleus RNA-sequencing identified compositional and transcriptional differences in adipocyte populations with age and cold exposure. Notably, cold exposure induced an adipocyte population expressing high levels of de novo lipogenesis (DNL) genes, and this response was severely blunted in aged animals. We further identified natriuretic peptide clearance receptor Npr3, a beige fat repressor, as a marker gene for a subset of white adipocytes and an aging-upregulated gene in adipocytes. In summary, this study indicates that aging blocks beige adipogenesis and dysregulates adipocyte responses to cold exposure and provides a unique resource for identifying cold and aging-regulated pathways in adipose tissue.

Control of lipolysis by a population of oxytocinergic sympathetic neurons.

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 ß-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.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

Objective: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. Methods: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice, to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active UCP1 expression. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. Results: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. However, endogenous Ucp1 was not actively expressed as Ucp1-CreERT2 failed to induce the reporter expression in the mammary glands. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. Conclusions: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

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

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

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

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

Palmitate Potentiates Lipopolysaccharide-Induced IL-6 Production via Coordinated Acetylation of H3K9/H3K18, p300, and RNA Polymerase II.

IL-6 is elevated in obese individuals and participates in the metabolic dysfunction associated with that condition. However, the mechanisms that promote IL-6 expression in obesity are incompletely understood. Because elevated levels of palmitate and LPS have been reported in obesity, we investigated whether these agents interact to potentiate IL-6 production. In this study, we report that LPS induces higher levels of IL-6 in human monocytes in the presence of palmitate. Notably, the priming effect of palmitate is associated with enhanced p300 binding and transcription factor recruitment to Il6 promoter regions. Gene silencing of p300 blocks this action of palmitate. RNA polymerase II recruitment was also enhanced at the Il6 promoter in palmitate/LPS-exposed cells. Acetylation levels of H3K9 and H3K18 were increased in monocytes treated with palmitate. Moreover, LPS stimulation of palmitate-treated cells led to increased levels of the transcriptionally permissive acetylation marks H3K9/H3K18 in the Il6 promoter compared with LPS alone. The effect of palmitate on LPS-induced IL-6 production was suppressed by the inhibition of histone acetyltransferases. Conversely, histone deacetylase inhibitors trichostatin A or sodium butyrate can substitute for palmitate in IL-6 production. Esterification of palmitate with CoA was involved, whereas ß-oxidation and ceramide biosynthesis were not required, for the induction of IL-6 and H3K9/H3K18 acetylation. Monocytes of obese individuals showed significantly higher H3K9/H3K18 acetylation and Il6 expression. Overall, our findings support a model in which increased levels of palmitate in obesity create a setting for LPS to potentiate IL-6 production via chromatin remodeling, enabling palmitate to contribute to metabolic inflammation.

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

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

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

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

Crosstalk Between Adipose and Lymphatics in Health and Disease.

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.

Isthmin-1 is an adipokine that promotes glucose uptake and improves glucose tolerance and hepatic steatosis.

With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

Mesothelial cells are not a source of adipocytes in mice.

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

Neurotensin is an anti-thermogenic peptide produced by lymphatic endothelial cells.

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.

IRF3 reduces adipose thermogenesis via ISG15-mediated reprogramming of glycolysis.

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.

Insulin-sensitizing effects of vitamin D repletion mediated by adipocyte vitamin D receptor: Studies in humans and mice.

OBJECTIVE: Adipose tissue inflammation and fibrosis appear to contribute to insulin resistance in obesity. Vitamin D receptor (Vdr) genes are expressed by adipocytes, macrophages, and fibroblasts, all of which could potentially play a role in adipose tissue inflammation and fibrosis. As vitamin D has been shown to have direct anti-inflammatory effects on adipocytes, we determined whether specific vitamin D receptor-mediated effects on adipocytes could impact adipose tissue inflammation and fibrosis and ultimately insulin resistance. METHODS: We examined the effects of repleting vitamin D in 25(OH)D-deficient, insulin resistant, overweight-to-obese human subjects (n = 19). A comprehensive assessment of whole-body insulin action was undertaken with stepped euglycemic (∼90 mg/dL) hyperinsulinemic clamp studies both before and after the administration of vitamin D or placebo. Adipose tissue fibrosis and inflammation were quantified by real-time rt-PCR and immunofluorescence in subcutaneous abdominal adipose tissue. To determine whether vitamin D's effects are mediated through adipocytes, we conducted hyperinsulinemic clamp studies (4 mU/kg/min) and adipose tissue analysis using an adipocyte-specific vitamin D receptor knockout (VDR-KO) mouse model (adiponectin-Cre + VDR+/fl) following high-fat diet feeding for 12 weeks. RESULTS: 25(OH)D repletion was associated with reductions in adipose tissue expression of pro-inflammatory and pro-fibrotic genes, decreased collagen immunofluorescence, and improved hepatic insulin sensitivity in humans. Worsening trends after six months on placebo suggest progressive metabolic effects of 25(OH)D deficiency. Ad-VDR-KO mice mirrored the vitamin D-deficient humans, displaying increased adipose tissue fibrosis and inflammation and hepatic insulin resistance. CONCLUSIONS: These complementary human and rodent studies support a beneficial role of vitamin D repletion for improving hepatic insulin resistance and reducing adipose tissue inflammation and fibrosis in targeted individuals, likely via direct effects on adipocytes. These studies have far-reaching implications for understanding the role of adipocytes in mediating adipose tissue inflammation and fibrosis and ultimately impacting insulin sensitivity.

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3.

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.