Extracellular metallothionein as a therapeutic target in the early progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by lymphocyte infiltration into the pancreatic islets of Langerhans, leading to the destruction of insulin-producing beta cells and uncontrolled hyperglycemia. In the nonobese diabetic (NOD) murine model of T1D, the onset of this infiltration starts several weeks before glucose dysregulation and overt diabetes. Recruitment of immune cells to the islets is mediated by several chemotactic cytokines, including CXCL10, while other cytokines, including SDF-1α, can confer protective effects. Global gene expression studies of the pancreas from prediabetic NOD mice and single-cell sequence analysis of human islets from prediabetic, autoantibody-positive patients showed an increased expression of metallothionein (MT), a small molecular weight, cysteine-rich metal-binding stress response protein. We have shown that beta cells can release MT into the extracellular environment, which can subsequently enhance the chemotactic response of Th1 cells to CXCL10 and interfere with the chemotactic response of Th2 cells to SDF-1α. These effects can be blocked in vitro with a monoclonal anti-MT antibody, clone UC1MT. When administered to NOD mice before the onset of diabetes, UC1MT significantly reduces the development of T1D. Manipulation of extracellular MT may be an important approach to preserving beta cell function and preventing the development of T1D.

Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.

BACKGROUND: Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT. METHODS: Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [Ucp1]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq (RNA sequencing) was performed in BAT from WT and Ucp1-/- mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in Bmp3b (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b. RESULTS: Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, Bmp3b-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to Bmp3b-deficient mice reduced I/R injury whereas transplanting BAT from Bmp3b-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury. CONCLUSIONS: The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System.

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

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.

Phosphatase protector alpha4 (α4) is involved in adipocyte maintenance and mitochondrial homeostasis through regulation of insulin signaling.

Insulin signaling is mediated via a network of protein phosphorylation. Dysregulation of this network is central to obesity, type 2 diabetes and metabolic syndrome. Here we investigate the role of phosphatase binding protein Alpha4 (α4) that is essential for the serine/threonine protein phosphatase 2A (PP2A) in insulin action/resistance in adipocytes. Unexpectedly, adipocyte-specific inactivation of α4 impairs insulin-induced Akt-mediated serine/threonine phosphorylation despite a decrease in the protein phosphatase 2A (PP2A) levels. Interestingly, loss of α4 also reduces insulin-induced insulin receptor tyrosine phosphorylation. This occurs through decreased association of α4 with Y-box protein 1, resulting in the enhancement of the tyrosine phosphatase protein tyrosine phosphatase 1B (PTP1B) expression. Moreover, adipocyte-specific knockout of α4 in male mice results in impaired adipogenesis and altered mitochondrial oxidation leading to increased inflammation, systemic insulin resistance, hepatosteatosis, islet hyperplasia, and impaired thermogenesis. Thus, the α4 /Y-box protein 1(YBX1)-mediated pathway of insulin receptor signaling is involved in maintaining insulin sensitivity, normal adipose tissue homeostasis and systemic metabolism.

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.

Brand interchangeability of pepsinogen tests in the real-world setting after eradication of Helicobacter pylori: a community-based study.

BACKGROUND: Serum pepsinogen (PG) is recommended as a screening test for premalignant gastric lesions. However, real-world evidence demonstrating its applicability and equivalence between different test brands is limited. METHODS: Mass screening began in 2018 in a high-risk Taiwanese population after eradication of Helicobacter pylori, with the first stage of two PG tests (GastroPanel®, Helsinki, Finland and LZ-Test®, Tokyo, Japan) and the second stage of endoscopy. A positive test was defined as PG-I < 30 ng/mL or PG-I/II ratio < 3 for GastroPanel® and PG-I ≤ 70 ng/mL and PG-I/II ratio ≤ 3 for LZ-Test®. Index lesions included atrophic gastritis and intestinal metaplasia. Test performance was evaluated based on the participation rate, positivity rate, referral rate, positive predictive value (PPV), and the detection rate. RESULTS: Among 7616 eligible participants, 5117 (67.2%) received PG tests and 284 (5.6%) tested positive. Of those who tested positive, 105 (37.0%) underwent endoscopy. Overall PPVs for atrophic gastritis and intestinal metaplasia were 12.4% and 18.9%, respectively, with detection rates of 2.5 and 3.9 per 1000, respectively. Correlations of numerical measures between tests were high and the agreements of test results were substantial. The PPVs (16.3% vs. 16.3% and 23.8% vs. 21.3%, P = 1.00 and 0.71, respectively), detection rates (2.5 vs. 2.5 and 3.7 vs. 3.3 per 1000, P = 1.00 and 0.27, respectively), and the stage distributions of gastritis were all comparable, which were confirmed by multiple regression analyses. CONCLUSIONS: PG testing is effective for mass screening after eradication of H. pylori. Tests from different manufacturers, even using different analytical methods and cutoff criteria, can perform equivalently.

Cold-induction of afadin in brown fat supports its thermogenic capacity.

The profound energy-expending nature of brown adipose tissue (BAT) thermogenesis makes it an attractive target tissue to combat obesity-associated metabolic disorders. While cold exposure is the strongest inducer of BAT activity, the temporal mechanisms tuning BAT adaptation during this activation process are incompletely understood. Here we show that the scaffold protein Afadin is dynamically regulated by cold in BAT, and participates in cold acclimation. Cold exposure acutely increases Afadin protein levels and its phosphorylation in BAT. Knockdown of Afadin in brown pre-adipocytes does not alter adipogenesis but restricts ß3-adrenegic induction of thermogenic genes expression and HSL phosphorylation in mature brown adipocytes. Consistent with a defect in thermogenesis, an impaired cold tolerance was observed in fat-specific Afadin knockout mice. However, while Afadin depletion led to reduced Ucp1 mRNA induction by cold, stimulation of Ucp1 protein was conserved. Transcriptomic analysis revealed that fat-specific ablation of Afadin led to decreased functional enrichment of gene sets controlling essential metabolic functions at thermoneutrality in BAT, whereas it led to an altered reprogramming in response to cold, with enhanced enrichment of different pathways related to metabolism and remodeling. Collectively, we demonstrate a role for Afadin in supporting the adrenergic response in brown adipocytes and BAT function.

Vascular smooth muscle-derived Trpv1+ progenitors are a source of cold-induced thermogenic adipocytes.

Brown adipose tissue (BAT) and beige fat function in energy expenditure in part due to their role in thermoregulation, making these tissues attractive targets for treating obesity and metabolic disorders. While prolonged cold exposure promotes de novo recruitment of brown adipocytes, the exact sources of cold-induced thermogenic adipocytes are not completely understood. Here, we identify transient receptor potential cation channel subfamily V member 1 (Trpv1)+ vascular smooth muscle (VSM) cells as previously unidentified thermogenic adipocyte progenitors. Single-cell RNA sequencing analysis of interscapular brown adipose depots reveals, in addition to the previously known platelet-derived growth factor receptor (Pdgfr)α-expressing mesenchymal progenitors, a population of VSM-derived adipocyte progenitor cells (VSM-APC) expressing the temperature-sensitive cation channel Trpv1. We demonstrate that cold exposure induces the proliferation of Trpv1+ VSM-APCs and enahnces their differentiation to highly thermogenic adipocytes. Together, these findings illustrate the landscape of the thermogenic adipose niche at single-cell resolution and identify a new cellular origin for the development of brown and beige adipocytes.

Brown Fat-Activating Lipokine 12,13-diHOME in Human Milk Is Associated With Infant Adiposity.

CONTEXT: Little is known about the specific breastmilk components responsible for protective effects on infant obesity. Whether 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), an oxidized linoleic acid metabolite and activator of brown fat metabolism, is present in human milk, or linked to infant adiposity, is unknown. OBJECTIVE: To examine associations between concentrations of 12,13-diHOME in human milk and infant adiposity. DESIGN: Prospective cohort study from 2015 to 2019, following participants from birth to 6 months of age. SETTING: Academic medical centers. PARTICIPANTS: Volunteer sample of 58 exclusively breastfeeding mother-infant pairs; exclusion criteria included smoking, gestational diabetes, and health conditions with the potential to influence maternal or infant weight gain. MAIN OUTCOME MEASURES: Infant anthropometric measures including weight, length, body mass index (BMI), and body composition at birth and at 1, 3, and 6 months postpartum. RESULTS: We report for the first time that 12,13-diHOME is present in human milk. Higher milk 12,13-diHOME level was associated with increased weight-for-length Z-score at birth (ß = 0.5742, P = 0.0008), lower infant fat mass at 1 month (P = 0.021), and reduced gain in BMI Z-score from 0 to 6 months (ß = -0.3997, P = 0.025). We observed similar associations between infant adiposity and milk abundance of related oxidized linoleic acid metabolites 12,13-Epoxy-9(Z)-octadecenoic acid (12,13-epOME) and 9,10-Dihydroxy-12-octadecenoic acid (9,10-diHOME), and metabolites linked to thermogenesis including succinate and lyso-phosphatidylglycerol 18:0. Milk abundance of 12,13-diHOME was not associated with maternal BMI, but was positively associated with maternal height, milk glucose concentration, and was significantly increased after a bout of moderate exercise. CONCLUSIONS: We report novel associations between milk abundance of 12,13-diHOME and adiposity during infancy.

The Link between Stress and IL-6 Is Heating Up.

Psychological stress has long been known to reduce adaptability to inflammatory challenges, although the precise mechanism has remained elusive. In a recent issue of Cell, Qing et al. (2020) demonstrate that psychological stress induces secretion of IL-6 from brown adipose tissue, which promotes hepatic gluconeogenesis, and reduces host fitness to inflammatory insults.

The Perlman syndrome DIS3L2 exoribonuclease safeguards endoplasmic reticulum-targeted mRNA translation and calcium ion homeostasis.

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.

FGF6 and FGF9 regulate UCP1 expression independent of brown adipogenesis.

Uncoupling protein-1 (UCP1) plays a central role in energy dissipation in brown adipose tissue (BAT). Using high-throughput library screening of secreted peptides, we identify two fibroblast growth factors (FGF), FGF6 and FGF9, as potent inducers of UCP1 expression in adipocytes and preadipocytes. Surprisingly, this occurs through a mechanism independent of adipogenesis and involves FGF receptor-3 (FGFR3), prostaglandin-E2 and interaction between estrogen receptor-related alpha, flightless-1 (FLII) and leucine-rich-repeat-(in FLII)-interacting-protein-1 as a regulatory complex for UCP1 transcription. Physiologically, FGF6/9 expression in adipose is upregulated by exercise and cold in mice, and FGF9/FGFR3 expression in human neck fat is significantly associated with UCP1 expression. Loss of FGF9 impairs BAT thermogenesis. In vivo administration of FGF9 increases UCP1 expression and thermogenic capacity. Thus, FGF6 and FGF9 are adipokines that can regulate UCP1 through a transcriptional network that is dissociated from brown adipogenesis, and act to modulate systemic energy metabolism.

TGF-ß2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism.

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-ß2 (TGF-ß2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-ß2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-ß2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2-/- mice, into sedentary mice improves glucose tolerance. TGF-ß2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-ß2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-ß2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-ß2-signaling cycle.

Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation.

OBJECTIVES: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. METHODS: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs. RESULTS: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development. CONCLUSION: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Deciphering adipose tissue heterogeneity.

Obesity is an excess accumulation of adipose tissue mass, and, together with its sequelae, in particular type II diabetes and metabolic syndrome, obesity presents a major health crisis. Although obesity is simply caused by increased adipose mass, the heterogeneity of adipose tissue in humans means that the response to increased energy balance is highly complex. Individual subjects with similar phenotypes may respond very differently to the same treatments; therefore, obesity may benefit from a personalized precision medicine approach. The variability in the development of obesity is indeed driven by differences in sex, genetics, and environment, but also by the various types of adipose tissue as well as the different cell types that compose it. By describing the distinct cell populations that reside in different fat depots, we can interpret the complex effect of these various players in the maintenance of whole-body energy homeostasis. To further understand adipose tissue, adipogenic differentiation and the transcriptional program of lipid accumulation must be investigated. As the cell- and depot-specific functions are described, they can be placed in the context of energy excess to understand how the heterogeneity of adipose tissue shapes individual metabolic status and condition.

Loss of BMP receptor type 1A in murine adipose tissue attenuates age-related onset of insulin resistance.

AIMS/HYPOTHESIS: Adipose tissue dysfunction is a prime risk factor for the development of metabolic disease. Bone morphogenetic proteins (BMPs) have previously been implicated in adipocyte formation. Here, we investigate the role of BMP signalling in adipose tissue health and systemic glucose homeostasis. METHODS: We employed the Cre/loxP system to generate mouse models with conditional ablation of BMP receptor 1A in differentiating and mature adipocytes, as well as tissue-resident myeloid cells. Metabolic variables were assessed by glucose and insulin tolerance testing, insulin-stimulated glucose uptake and gene expression analysis. RESULTS: Conditional deletion of Bmpr1a using the aP2 (also known as Fabp4)-Cre strain resulted in a complex phenotype. Knockout mice were clearly resistant to age-related impairment of insulin sensitivity during normal and high-fat-diet feeding and showed significantly improved insulin-stimulated glucose uptake in brown adipose tissue and skeletal muscle. Moreover, knockouts displayed significant reduction of variables of adipose tissue inflammation. Deletion of Bmpr1a in myeloid cells had no impact on insulin sensitivity, while ablation of Bmpr1a in mature adipocytes partially recapitulated the initial phenotype from aP2-Cre driven deletion. Co-cultivation of macrophages with pre-adipocytes lacking Bmpr1a markedly reduced expression of proinflammatory genes. CONCLUSIONS/INTERPRETATION: Our findings show that altered BMP signalling in adipose tissue affects the tissue's metabolic properties and systemic insulin resistance by altering the pattern of immune cell infiltration. The phenotype is due to ablation of Bmpr1a specifically in pre-adipocytes and maturing adipocytes rather than an immune cell-autonomous effect. Mechanistically, we provide evidence for a BMP-mediated direct crosstalk between pre-adipocytes and macrophages.

Genetic and functional characterization of clonally derived adult human brown adipocytes.

Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and its activation to a state of increased energy expenditure is believed to protect against the development of obesity. Even though the existence of BAT in adult humans has been widely appreciated, its cellular origin and molecular identity remain elusive largely because of high cellular heterogeneity within various adipose tissue depots. To understand the nature of adult human brown adipocytes at single cell resolution, we isolated clonally derived adipocytes from stromal vascular fractions of adult human BAT from two individuals and globally analyzed their molecular signatures. We used RNA sequencing followed by unbiased genome-wide expression analyses and found that a population of uncoupling protein 1 (UCP1)-positive human adipocytes possessed molecular signatures resembling those of a recruitable form of thermogenic adipocytes (that is, beige adipocytes). In addition, we identified molecular markers that were highly enriched in UCP1-positive human adipocytes, a set that included potassium channel K3 (KCNK3) and mitochondrial tumor suppressor 1 (MTUS1). Further, we functionally characterized these two markers using a loss-of-function approach and found that KCNK3 and MTUS1 were required for beige adipocyte differentiation and thermogenic function. The results of this study present new opportunities for human BAT research, such as facilitating cell-based disease modeling and unbiased screens for thermogenic regulators.