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SummaryBrown adipose tissue (BAT) has the capacity to regulate systemic metabolism through the secretion of signaling lipids. N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional mRNA modification and has been reported to regulate BAT adipogenesis and energy expenditure. In this study, we demonstrate that the absence of m6A methyltransferase-like 14 (METTL14), modifies the BAT secretome to initiate inter-organ communication to improve systemic insulin sensitivity. Importantly, these phenotypes are independent of UCP1-mediated energy expenditure and thermogenesis. Using lipidomics, we identified prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) as M14KO-BAT-secreted insulin sensitizers. Notably, circulatory PGE2 and PGF2a levels are inversely correlated with insulin sensitivity in humans. Furthermore,in vivoadministration of PGE2 and PGF2a in high-fat diet-induced insulin-resistant obese mice recapitulates the phenotypes of METTL14 deficient animals. PGE2 or PGF2a improves insulin signaling by suppressing the expression of specific AKT phosphatases. Mechanistically, METTL14-mediated m6A installation promotes decay of transcripts encoding prostaglandin synthases and their regulators in human and mouse brown adipocytes in a YTHDF2/3-dependent manner. Taken together, these findings reveal a novel biological mechanism through which m6A-dependent regulation of BAT secretome regulates systemic insulin sensitivity in mice and humans.HighlightsMettl14KO-BAT improves systemic insulin sensitivity via inter-organ communication;PGE2 and PGF2a are BAT-secreted insulin sensitizers and browning inducers;PGE2 and PGF2a sensitize insulin responses through PGE2-EP-pAKT and PGF2a-FP-AKT axis;METTL14-mediated m6A installation selectively destabilizes prostaglandin synthases and their regulator transcripts;Targeting METTL14 in BAT has therapeutic potential to enhance systemic insulin sensitivityAbstract Figure

Obesity induces chronic inflammation resulting in insulin resistance and metabolic disorders. Cold exposure can improve insulin sensitivity in humans and rodents, but the mechanisms have not been fully elucidated. Here, we find that cold resolves obesity-induced inflammation and insulin resistance and improves glucose tolerance in diet-induced obese mice. The beneficial effects of cold exposure on improving obesity-induced inflammation and insulin resistance depend on brown adipose tissue (BAT) and liver. Using targeted liquid chromatography with tandem mass spectrometry, we discovered that cold and β3-adrenergic stimulation promote BAT to produce maresin 2 (MaR2), a member of the specialized pro-resolving mediators of bioactive lipids that play a role in the resolution of inflammation. Notably, MaR2 reduces inflammation in obesity in part by targeting macrophages in the liver. Thus, BAT-derived MaR2 could contribute to the beneficial effects of BAT activation in resolving obesity-induced inflammation and may inform therapeutic approaches to combat obesity and its complications.

Single-cell RNA sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue, for which collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nucleus extraction is often much more efficient and therefore single-nucleus RNA sequencing (snRNA-seq) presents an alternative to scRNA-seq. However, nuclear transcripts represent only a fraction of the transcriptome in a single cell, with snRNA-seq marked with inherent transcript enrichment and detection biases. Therefore, snRNA-seq may be inadequate for mapping important transcriptional signatures in adipose tissue. In this study, we compare the transcriptomic landscape of single nuclei isolated from preadipocytes and mature adipocytes across human white and brown adipocyte lineages, with whole-cell transcriptome. We show that snRNA-seq is capable of identifying the broad cell types present in scRNA-seq at all states of adipogenesis. However, we also explore how and why the nuclear transcriptome is biased and limited, as well as how it can be advantageous. We robustly characterize the enrichment of nuclear-localized transcripts and adipogenic regulatory lncRNAs in snRNA-seq, while also providing a detailed understanding for the preferential detection of long genes upon using this technique. To remove such technical detection biases, we propose a normalization strategy for a more accurate comparison of nuclear and cellular data. Finally, we show successful integration of scRNA-seq and snRNA-seq data sets with existing bioinformatic tools. Overall, our results illustrate the applicability of snRNA-seq for the characterization of cellular diversity in the adipose tissue.

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis

BACKGROUND: Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS: We treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m(2)) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [(18)F]-2-fluoro-d-2-deoxy-d-glucose ((18)F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test. RESULTS: Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION: These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION: Clinicaltrials.gov NCT03049462. FUNDING: This work was supported by grants from the Intramural Research Program of the NIDDK, NIH (DK075112, DK075116, DK071013, and DK071014).

The current obesity pandemic results from a physiological imbalance in which energy intake chronically exceeds energy expenditure (EE), and prevention and treatment strategies remain generally ineffective. Approaches designed to increase EE have been informed by decades of experiments in rodent models designed to stimulate adaptive thermogenesis, a long-term increase in metabolism, primarily induced by chronic cold exposure. At the cellular level, thermogenesis is achieved through increased rates of futile cycling, which are observed in several systems, most notably the regulated uncoupling of oxidative phosphorylation from ATP generation by uncoupling protein 1, a tissue-specific protein present in mitochondria of brown adipose tissue (BAT). Physiological activation of BAT and other organ thermogenesis occurs through β-adrenergic receptors (AR), and considerable effort over the past 5 decades has been directed toward developing AR agonists capable of safely achieving a net negative energy balance while avoiding unwanted cardiovascular side effects. Recent discoveries of other BAT futile cycles based on creatine and succinate have provided additional targets. Complicating the current and developing pharmacological-, cold-, and exercise-based methods to increase EE is the emerging evidence for strong physiological drives toward restoring lost weight over the long term. Future studies will need to address technical challenges such as how to accurately measure individual tissue thermogenesis in humans; how to safely activate BAT and other organ thermogenesis; and how to sustain a negative energy balance over many years of treatment.

An appealing strategy for treatment of metabolic disease in humans is activation of brown adipose tissue (BAT), a thermogenic organ best visualized through

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of β-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and β3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A–Gαq–futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.

Bile acids (BAs) are classically known as facilitators of fat digestion. Moreover, they are now known to be ligands for receptors involved in glucose and lipoprotein metabolism. BA synthesis was originally thought to be exclusive to hepatic tissue, but emerging evidence in rodents has demonstrated the expression of BA transporters and de novo synthetic enzymes in murine adipose tissue. We previously showed that chronic activation of brown adipose tissue (BAT) and white adipose tissue (WAT) in adult women via the β3-adrenergic receptor (ADRB3) agonist mirabegron increases total plasma BA levels. The largest increases were seen with primary unconjugated BAs. Overall, these data suggest that chronic adrenergic stimulation may increase the synthesis and activity of BA synthesis enzymes in human adipose tissue, which contributes to the increase in total plasma BAs. To confirm this hypothesis, we measured expression levels of BAT and BA synthesis enzymes in liver and adipose tissues collected from healthy patients and those with pheochromocytoma (pheo) who experienced chronic adrenergic activation. As expected, expression of thermogenic UCP1 and ADRB3 was higher in pheo peri-renal BAT than in liver tissue (76,000x, P0.05), and liver had slightly higher expression than BAT (3x, P=0.03). Otherwise, most of the principal BA synthesis genes’ expression levels demonstrated the following stepwise pattern: liver tissue > pheo WAT = subcutaneous WAT > BAT (CYP8B1, CYP27A1, AKR1D1, and BAAT). These results suggest that BA production in humans is not limited to the liver. Rather, human WAT in particular may contribute to the BA pool and could serve as a target for producing BA-mediated improvements in glucose and lipoprotein metabolism. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Background Obesity results from excess adipose triglyceride storage and leads to multiple life-threatening complications that may be amenable to novel pharmacological therapies. Both human white and brown adipocytes express β1-, β2-, and β3-adrenergic receptors (β-AR's), and stimulation of each activates converging signaling pathways that regulate essential metabolic and cellular functions. Clinical data indicate that acute and chronic stimulation of the β3-AR with the selective agonist mirabegron increases lipolysis and thermogenesis, thereby making β3-AR activation a promising target to improve obesity-related metabolic disease. Purpose In this study, we evaluated in vitro the short-term and prolonged activation of β3-ARs via mirabegron and compared the effects to the activation of β1- and β2-AR. Methods Functional cellular assays such as lipolysis and cellular respiration, were measured in immortalized human white (hWA) and brown (hBA) adipocytes derived from the superficial and deep neck depots, respectively. Cells were treated for 6 h (short-term) and 24 h (prolonged) with mirabegron (β3-AR agonist), dobutamine (β1-AR agonist), terbutaline (β2-AR agonist) and isoproterenol (non-selective β-AR agonist). One-way ANOVA was used for statistical analysis. Findings In hWA, only short-term activation of the β2-AR induced lipolysis (1.8-fold increase, P=0.0002), while in hBA, both the β2-AR (1.5-fold increase, P=0.04) and β3-AR (1.6 fold increase, P=0.002) increased lipolysis with 6h treatment, as compared to the vehicle. By 24h, lipolysis was blunted in both cell types, likely due to agonist-induced desensitization and downregulation. Distinct from the lipolytic effects ofβ-AR activation, the basal oxygen consumption rate (OCR) was not significantly different among hBA and hWA treated acutely and chronically with each of the three selective β-ARs and positive control isoproterenol. However, in hBA only, prolonged activation of all three β-AR's lowered maximal mitochondrial respiration (isoproterenol: 25% decrease, P=0.0004; dobutamine: 25% decrease, P=0.0003; terbutaline: 26% decrease, P=0.0004; mirabegron: 38% decrease, P Conclusion In hBA, acute stimulation of either the β2-AR or β3-AR induces lipolysis. Prolonged exposure to β-agonists comparably lowers maximal cellular respiration, suggesting that all β-AR's are subject to agonist-induced desensitization. Moreover, continuous activation of lipolysis may deplete the intracellular fatty acid pool necessary for mitochondrial oxidative respiration. These responses will need to be addressed before novel adrenergic activators can be utilized to reduce triglyceride stores and treat obesity-related metabolic disease. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:00 p.m. - 1:05 p.m.

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.

Context Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumors that can secrete norepinephrine (NE). Brown adipose tissue (BAT) activation is mediated through the action of NE on β-adrenoceptors (β-ARs). In some malignancies, BAT activation is associated with higher cancer activity. Objective To study the relationship between BAT activation and PPGL clinical outcomes. Design A retrospective case-control study that included 342 patients with PPGLs who underwent 18F-fluoro-2-deoxy-D-glucose positron emission tomography-computed tomography (18F-FDG PET/CT) imaging at the National Institutes of Health (NIH). We excluded all patients with parasympathetic tumors and those who underwent 18F-FDG PET/CT after PPGL resection. Scans of 205 patients were reviewed by 2 blinded nuclear medicine physicians; 16 patients had BAT activation on 18F-FDG PET/CT [7.80%; age 27.50 (15.00–45.50) years; 10 female/6 male; body mass index [BMI] 24.90 [19.60–25.35] kg/m2). From the remaining 189 patients, we selected 36 matched controls (age 34.4 [25.4–45.5] years; 21 female/15 male; BMI 25.0 [22.0–26.0] kg/m2). Primary Outcome Measure Overall survival. Results The presence of active BAT on 18F-FDG PET/CT was associated with decreased overall survival when compared with the control group (HRz 5.80; 95% CI, 1.05–32.05; P = 0.02). This association remained significant after adjusting for the SDHB mutation. Median plasma NE in the BAT group was higher than the control group [4.65 vs 0.55 times above the upper limit of normal; P < 0.01]. There was a significant association between higher plasma NE levels and mortality in PPGLs in both groups. Conclusions Our findings suggest that the detection of BAT activity in PPGL patients is associated with higher mortality. We suggest that BAT activation could either be reflecting or contributing to a state of increased host stress that may predict poor outcome in metastatic PPGL.

Context Patients with mutations of the insulin receptor gene (INSR) have extreme insulin resistance and are at risk for early morbidity and mortality from diabetes complications. A case report suggested that thyroid hormone could improve glycemia in INSR mutation in part by increasing brown adipose tissue (BAT) activity and volume. Objective To determine if thyroid hormone increases tissue glucose uptake and improves hyperglycemia in INSR mutation. Design Single-arm, open-label study of liothyronine. Setting National Institutes of Health. Participants Patients with homozygous (n = 5) or heterozygous (n = 2) INSR mutation. Intervention Liothyronine every 8 hours for 2 weeks (n = 7); additional 6 months’ treatment in those with hemoglobin A1c (HbA1c) > 7% (n = 4). Outcomes Whole-body glucose uptake by isotopic tracers; tissue glucose uptake in muscle, white adipose tissue (WAT) and BAT by dynamic [18F] fluorodeoxyglucose positron emission tomography/computed tomography; HbA1c. Results There was no change in whole-body, muscle, or WAT glucose uptake from baseline to 2 weeks of liothyronine. After 6 months, there was no change in HbA1c (8.3 ± 1.2 vs 9.1 ± 3.0%, P = 0.27), but there was increased whole-body glucose disposal (22.8 ± 4.9 vs 30.1 ± 10.0 µmol/kg lean body mass/min, P = 0.02), and muscle (0.7 ± 0.1 vs 2.0 ± 0.2 µmol/min/100 mL, P < 0.0001) and WAT glucose uptake (1.2 ± 0.2 vs 2.2 ± 0.3 µmol/min/100 mL, P < 0.0001). BAT glucose uptake could not be quantified because of small volume. There were no signs or symptoms of hyperthyroidism. Conclusion Liothyronine administered at well-tolerated doses did not improve HbA1c. However, the observed increases in muscle and WAT glucose uptake support the proposed mechanism that liothyronine increases tissue glucose uptake. More selective agents may be effective at increasing tissue glucose uptake without thyroid hormone–related systemic toxicity. Clinical Trial Registration Number: NCT02457897; https://clinicaltrials.gov/ct2/show/NCT02457897.

Cushing Syndrome (CS) is characterized by cortisol excess that leads to impaired glucose tolerance and obesity. CT imaging shows 20% of CS patients have nonalcoholic steatohepatitis (NASH). While the gold standard diagnostic test for NASH is liver biopsy, it has too much risk for CS patients. This study evaluated the ability of noninvasive, magnetic resonance spectroscopy (MRS) to measure liver fat percentage in CS patients and followed temporal changes in metabolic parameters before and after successful treatment. The primary outcome was protein density fat fraction (PDFF) by MRS at 3 timepoints: baseline, 6 months, and 12 months after treatment, which consisted of surgery as indicated based on ACTH source. The diagnosis was confirmed if there was histology consistent with corticotrope adenoma, adrenal tumor, or an ectopic source. We prospectively studied 41 consecutive patients: 85% female; 44±1.8 y; 32.6±1.5 kg/m²; urine cortisol excretion 2242.7±1806.3 [nl 3.5-45.0 mcg/24h]). Each underwent MRS at 3T where NASH was defined as >5% PDFF. A1c and BMI were measured. Changes over time were evaluated using Wilcoxon signed-rank test, and correlation among variables was done with Spearman’s rank test. At baseline, mean PDFF was 10.4±1.7 and correlated positively with BMI (r=0.57, P In summary, MRS-PDFF was effective at diagnosing NASH in CS. Physiologically, definitive treatment of CS reduced liver fat by 6 months after normalization of cortisol and also preceded the improvement in A1c. These results suggest that liver insulin resistance due to fat accumulation has an important role in diabetes pathophysiology in CS. Disclosure A. Pierce: None. Z. A. Sater: None. A. M. Cypess: None.

Objective Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. Methods LDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the β3-adrenergic receptor (β3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. Results Reducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. Conclusions Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT., Graphical abstract Image 1, Highlights • The transcriptional co-regulator LDB1 is required for brown adipocyte gene expression, including Ucp1. • Several LIM-domain factors, including Lmo2, Lmo4, and Lhx8, are expressed in BAT and may be potential LDB1 partners. • Male Ldb1 BAT knockouts are glucose and insulin intolerant, have lower glucose uptake and altered insulin signaling. • LDB1 impacts brown adipocyte morphology, lipid droplet size, and mRNA associated with lipid utilization. • BAT-expressed LDB1 is required for energy expenditure and cold tolerance.

Single-cell RNA-sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue where collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nuclei extraction is often much more efficient and therefore single-nuclei RNA-sequencing (snRNA-seq) presents an alternative to scRNA-seq. However, nuclear transcripts represent only a fraction of the transcriptome in a single cell, with snRNA-seq marked with inherent transcript enrichment and detection biases. Therefore, snRNA-seq may be inadequate for mapping important transcriptional signatures in adipose tissue. In this study, we compare the transcriptomic landscape of single nuclei isolated from preadipocytes and mature adipocytes across human white and brown adipocyte lineages, with whole-cell transcriptome. We demonstrate that snRNA-seq is capable of identifying the broad cell types present in scRNA-seq at all states of adipogenesis. However, we also explore how and why the nuclear transcriptome is biased and limited, and how it can be advantageous. We robustly characterize the enrichment of nuclear-localized transcripts and adipogenic regulatory lncRNAs in snRNA-seq, while also providing a detailed understanding for the preferential detection of long genes upon using this technique. To remove such technical detection biases, we propose a normalization strategy for a more accurate comparison of nuclear and cellular data. Finally, we demonstrate successful integration of scRNA-seq and snRNA-seq datasets with existing bioinformatic tools. Overall, our results illustrate the applicability of snRNA-seq for characterization of cellular diversity in the adipose tissue.

Background Activation of brown adipose tissue (BAT) in rodents increases lipolysis in white adipose tissue (WAT) and improves glucose tolerance. Adult humans can have metabolically active BAT. Implications for diabetes and obesity in humans require a better characterization of BAT in humans. Purpose To study fat depots with localized proton MR spectroscopy relaxometry and to identify differences between WAT and fluorine 18 fluorodeoxyglucose (FDG) PET/CT proven cold-activated BAT in humans. Materials and Methods Participants were consecutively enrolled in this prospective study (ClinicalTrials.gov identifiers: NCT01568671 and NCT01399385) from August 2016 to May 2019. Supraclavicular potential BAT regions were localized with MRI. Proton densities, T1, and T2 were measured with localized MR spectroscopy in potential BAT and in subcutaneous WAT. FDG PET/CT after cold stimulation was used to retrospectively identify active supraclavicular BAT or supraclavicular quiescent adipose tissue (QAT) regions. MR spectroscopy results from BAT and WAT were compared with grouped and paired tests. Results Of 21 healthy participants (mean age, 36 years ± 16 [standard deviation]; 13 men) FDG PET/CT showed active BAT in 24 MR spectroscopy-targeted regions in 16 participants (eight men). Four men had QAT. The T2 for methylene protons was shorter in BAT (mean, 69 msec ± 6, 24 regions) than in WAT (mean, 83 msec ± 3, 18 regions, P < .01) and QAT (mean, 78 msec ± 2, five regions, P < .01). A T2 cut-off value of 76 msec enabled the differentiation of BAT from WAT or QAT with a sensitivity of 85% and a specificity of 95%. Densities of protons adjacent and between double bonds were 33% and 24% lower, respectively, in BAT compared with those in WAT (P = .01 and P = .03, respectively), indicating a lower content of unsaturated and polyunsaturated fatty acids, respectively, in BAT compared with WAT. Conclusion Proton MR spectroscopy showed shorter T2 and lower unsaturated fatty acids in brown adipose tissue (BAT) than that in white adipose tissue in healthy humans. It was feasible to identify BAT with MR spectroscopy without the use of PET/CT or cold stimulation. © RSNA, 2021 See also the editorial by Barker in this issue. Online supplemental material is available for this article.

RationaleHistone deacetylases (HDACs) play a central role in cardiac hypertrophy and fibrosis in preclinical models. However, their impact in the human heart remains unknown.ObjectiveWe aimed to image HDAC expression in the human heart in vivo with PET-MR (positron emission tomography and magnetic resonance) using [11C]Martinostat, a novel radiotracer targeted to class I HDACs. We further aimed to compare HDAC expression in the heart with its expression in skeletal muscle and brown/white adipose tissue (BAT/WAT).Methods and ResultsThe specificity and selectivity of [11C]Martinostat binding in the heart was assessed in non-human primates (n=2) by in vivo blocking studies and with an ex vivo cellular thermal shift assay (CETSA) of HDAC paralog stabilization by Martinostat. PET-MR imaging of [11C]Martinostat was performed in healthy volunteers (n=6) for 60 minutes to obtain time-activity curves of probe uptake and kinetics. qPCR of class I HDACs was performed in specimens of BAT obtained from patients (n=7) undergoing abdominal surgery and in specimens of human subcutaneous WAT (n=7). CETSA and the blocking studies demonstrated that Martinostat was specific for class I HDACs in the heart. HDAC density, measured by standardized uptake values of [11C]Martinostat, was 8 times higher in the myocardium than skeletal muscle (4.4 ± 0.6 vs. 0.54 ± 0.29, pConclusionsClass I HDAC expression in the human heart can be imaged in vivo and is dramatically higher than any other peripheral tissue, including skeletal muscle. The high levels of HDAC in the myocardium and BAT suggest that epigenetic regulation plays an important role in tissues with high energetic demands and metabolic plasticity.

Background We recently demonstrated that thrifty subjects, characterized by a greater decrease in 24 h energy expenditure (24hEE) during short-term fasting, have less capacity for cold-induced thermogenesis (CIT) during 24 h of mild cold exposure. Objective As cold-induced brown adipose tissue activation (CIBA) is a determinant of CIT, we sought to investigate whether thrifty individuals also have reduced CIBA. Methods Twenty-four healthy subjects (age: 29.8 ± 9.5y, body fat: 27.3 ± 12.4%, 63% male) were admitted to our clinical research unit and underwent two 24hEE assessments in a whole-room indirect calorimeter during energy balance and fasting conditions at thermoneutrality to quantify their degree of thriftiness. Positron emission tomography/computed tomography scans were performed after exposure to 16 °C for 2 h to quantify peak CIBA. Results A greater decrease in 24hEE during fasting was associated with lower peak CIBA (r = 0.50, p = 0.01), such that a 100 kcal/day greater reduction in 24hEE related to an average 3.2 g/mL lower peak CIBA. Conclusion Our results indicate that reduced CIBA is a metabolic trait of the thrifty phenotype which might explain reduced CIT capacity and greater predisposition towards weight gain in individuals with a thrifty metabolism.

Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. While increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Using single cell RNA sequencing of the stromal vascular fraction of murine BAT and analysis of 67 brown preadipocyte and adipocyte clones we unravel heterogeneity within brown preadipocytes. Statistical analysis of gene expression profiles from these clones identifies markers distinguishing brown adipocyte lineages. We confirm the presence of distinct brown adipocyte populationsin vivousing three identified markers; Eif5, Tcf25, and Bin1. Functionally, we demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that Bin1 marks a dormant brown adipocyte type. The existence of multiple brown adipocyte lineages suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct function in thermogenesis and the regulation of whole body energy homeostasis.

Beyond enabling digestion of fats, bile acids (BA) are now known to be receptor ligands that improve glucose and lipoprotein metabolism. Classically, regulation of BA synthesis has thought to be exclusively hepatic, but emerging evidence in rodents indicates that BA production is influenced by sympathetic activation of brown and white adipose tissue (BAT and WAT). We recently showed that chronic activation of BAT and WAT in adult women with the β3-adrenergic receptor (AR) agonist mirabegron increased HDL cholesterol, insulin sensitivity, and total plasma BA. The largest increases were with the primary unconjugated cholic and chenodeoxycholic acids. We hypothesized that chronic adrenergic stimulation increased BA synthesis enzymes in adipose tissue, thus increasing flux through these pathways. We therefore measured expression levels of BA synthesis enzymes from patients undergoing elective surgery in the following tissues: subcutaneous WAT (scWAT), and from pheochromocytoma (pheo) patients who have chronic adrenergic activation, scWAT (PhW) and peri-renal BAT (PhB). These were compared to cadaveric liver. Liver had higher expression levels of BAAT, which conjugates BA. However, expression levels among liver, scWAT, PhW, and PhB were similar for the enzymes initiating BA synthesis (CYP7A1) and other intermediate enzymes (CYP7B1, AKR1D1, AMCAR). Furthermore, pheo patients had higher levels of CYP27A1, which initiates the acidic/alternative BA synthesis pathway. These data suggest that regulation of plasma BA is not exclusive to the liver. Rather, human BAT and WAT likely contribute to the BA pool, and chronic adrenergic activation may shift the equilibrium toward primary unconjugated species to improve glucose and lipoprotein metabolism. Disclosure K.Y. Zhu: None. H.J. Lea: None. C. Cero: None. A.M. Cypess: None.

Recent clinical trials show that chronic activation of the β3-adrenergic receptor (AR) by mirabegron leads to numerous beneficial physiological responses, including increased energy expenditure, insulin sensitivity, and improved lipoprotein profile. Preliminary evidence suggests that they are mediated in part through adaptations by adipose tissue, but little is known about how mirabegron affects the cellular metabolism of human white or brown adipocytes (hWA and BA, respectively). We therefore treated paired immortalized hWA and hBA for 6 hours with a range of mirabegron doses (1 nM - 10 µM), as well as the β1- and β2-AR selective agonists, dobutamine and terbutaline, at 10 µM. We found that in untreated cells, ADRB1 and ADRB2 were higher in hWA than hBA, but ADRB3 was higher in hBA. However, mirabegron at all concentrations increased the expression of ADRB3 in hWA, and the highest dose of mirabegron boosted expression above that in hBA. While an increase in ADRB3 was detected in hWA with β1- and β2-agonist treatment, no changes occurred to the other two β-ARs when exposed to their selective agonists. Downstream, the β1- and β2-agonists increased thermogenic UCP1 expression in hWA, but mirabegron raised UCP1 expression only in hBA. Among the genes associated with lipolysis, glucose metabolism, and insulin signaling, mirabegron 10 µM promoted the expression of ATGL and HSL encoding genes, as well as GLUT4 in hBA only. In contrast, no changes occurred in hWA. In summary, chronic exposure to a β3-AR agonist primes human white adipocytes to respond more potently to adrenergic-mediated lipolysis and enables human brown adipocytes to increase their thermogenic capacity. Disclosure H.J. Lea: None. K.Y. Zhu: None. C. Cero: None. A.M. Cypess: None.

Activation of rodent brown adipose tissue (BAT) via pharmacological stimulation of β3-adrenergic receptors (β3-AR) leads to metabolic benefit, however in humans, the physiological relevance of BAT and β3-AR remain controversial. To investigate the role of the β3-AR in human brown adipocytes, first, we established a human primary supraclavicular adipocyte in vitro model that upon differentiation preserved the physiological properties brown adipose tissue with increased β-ARs, adipogenic, thermogenic and brown/beige markers. Next, we selective silenced β3-AR expression in differentiated primary brown/beige adipocytes using a lipofectamine-based non-viral transfection. 48h of transfection of siRNA targeting ADRB3 (siRNA-ADRB3) resulted in a 90% reduction in ADRB3 mRNA levels, relative to control siRNA-transfected cells (siRNA-Ctrl). siRNA-ADRB3 also reduced genes of fatty acid metabolism, thermogenesis (UCP1, 90% decrease), and mitochondrial mass. Functionally, lower cAMP levels, lipolysis, oxygen consumption rates and higher glycolysis were caused by silencing β3-AR receptor. Immortalized brown adipocyte (hBA’s), from a separate subject, displayed a similar decrease in lipolysis and cellular respiration. Mirabegron, the selective human β3-AR agonist, stimulated BAT lipolysis and thermogenesis, that were lost after knockdown ADRB3. Similarly, selective subtype human β-AR agonists dobutamine (β1-AR agonist) and terbutaline (β2-AR agonist) treatment also increased lipolysis and thermogenesis, that were also attenuated when β3-AR was silenced. Thus, the β3-AR maintains multiple components of the human brown/beige adipocyte lipolytic and thermogenic cellular machinery. These findings indicate that ADRB3 and β3-AR agonists could be used to achieve metabolic benefit in humans. Disclosure C. Cero: None. H.J. Lea: None. K.Y. Zhu: None. A.M. Cypess: None.

Increasing biological thermogenesis (the conversion of chemical energy to heat) has been proposed as an approach towards lowering blood glucose, improving insulin sensitivity and mitigating type 2 diabetes. Non-shivering thermogenesis occurs primarily in adipocytes that express uncoupling protein 1 (UCP1) including brown adipose tissue (BAT) and beige adipose tissue but does not substantially occur in white adipose tissue (WAT). To identify genes that regulate thermogenesis and BAT function, we recently compared genes expressed in paired human patient samples (n = 10) of the deep neck BAT located next to the carotid sheath and superficial subcutaneous WAT in the neck. We found UDP-glucuronide transferase 1A (UGT1A) is co-expressed in individuals with a high amount of the thermogenic protein uncoupling protein 1 (UCP1). This enzyme is known to glucuronidate metabolites, including bilirubin and xenobiotics, which increases their water solubility and excretion. Based on this function, we hypothesize UGT1A regulates BAT not through direct action of the enzyme but through modulating the availability of thermogenic molecules. Here, we characterized expression of different UGT1A isoforms in vivo and in vitro. Cold exposure is an effective method for activating BAT and increasing thermogenesis in mice. Interestingly, expression of multiple UGT1A isoforms, including UGT1A6a and UGT1A6b, are decreased in BAT of mice exposed to a cold environment (5°C) for 7 days. This finding is consistent with data in multiple publicly available datasets which have shown UGT1A6a/6b are decreased in cold exposure and other thermogenic stimuli. In vitro, most UGT1A isoforms were increased during the differentiation of murine brown adipocytes but only certain isoforms were differentially expressed during in vitro differentiation in human brown adipocytes. Based on these data, we propose that UGT1A may have a function related to thermogenesis and understanding its function could lead to novel approaches for activating BAT. Disclosure S. Kodani: None. M. Lynes: None. F. Shamsi: None. C. Wang: None. S. Sugimoto: None. R. Xue: None. T. Huang: None. A.P. White: None. A.M. Cypess: None. Y. Tseng: Consultant; Self; Boehringer Ingelheim International GmbH. Funding National Institutes of Health (R01DK07709, R01DK102898, T32DK007260)

Design and rationale: Obesity results from energy intake exceeding energy expenditure (EE) over a prolonged period. Many anti-obesity drugs are designed to decrease energy intake. However, their potential impact on EE is not well documented. We designed a placebo-controlled, double-blind, randomized cross-over study to determine the acute effects of several FDA-approved anti-obesity drugs on basal metabolic rate (BMR) under well-controlled conditions. Protocol and inclusion criteria: This ongoing study is limited to healthy males of all ethnicities aged 18–35 years with a BMI of 18.5 to 25.0 kg/m2. Following an overnight stay in the Metabolic Clinical Research Unit, fasting subjects were measured from 8:00am to 12:00pm in a whole-room indirect calorimeter, which was maintained at a thermoneutral temperature (26.7±0.9°C) to prevent non-shivering thermogenesis. The six treatments include placebo, caffeine as the positive control (300 mg), phentermine (37.5 mg), topiramate (200 mg), Qsymia (phentermine 15 mg / topiramate 92 mg), and naltrexone (100 mg), with a 1-week outpatient washout period after each treatment. Drug-naïve subjects received a single dose of each drug to minimize potential metabolic adaptations that may occur with weight-loss or chronic use. The prespecified primary outcome was a ≥5% increase in BMR vs. placebo for each drug. This difference can be detected for 16 subjects with 0.83 power at α=0.05 allowing for ≤25% dropout. Secondary outcomes include respiratory quotient (RQ), heart rate (HR), mean arterial pressure (MAP), and self-reported hunger. Preliminary data: To date, 7 subjects were recruited and 6 have completed the study (26.1±4.3 years, BMI 23.1±1.4 kg/m2, body fat percentage 18.4±4.1%). Interim analysis using paired t-tests shows, compared to placebo, caffeine trended towards increasing EE (1.17±0.07 vs. 1.27±0.12 kcal/min; p=0.07) and increased MAP by 5.5±4.2% (88±2 vs. 93±4; p Summary and future directions: Anti-obesity drugs may increase energy expenditure by upregulating sympathetic nervous system activity. Combined with appetite suppression, the impact on energy balance can lead to weight loss. We aim to complete our study to determine whether these drugs can acutely increase EE with minimal cardiovascular side-effects and compare our findings with long-term interventions.

β3-Adrenergic receptors (β3-ARs) are the predominant regulators of rodent brown adipose tissue (BAT) thermogenesis. However, in humans, the physiological relevance of BAT and β3-AR remains controversial. Herein, using primary human adipocytes from supraclavicular neck fat and immortalized brown/beige adipocytes from deep neck fat from 2 subjects, we demonstrate that the β3-AR plays a critical role in regulating lipolysis, glycolysis, and thermogenesis. Silencing of the β3-AR compromised genes essential for thermogenesis, fatty acid metabolism, and mitochondrial mass. Functionally, reduction of β3-AR lowered agonist-mediated increases in intracellular cAMP, lipolysis, and lipolysis-activated, uncoupling protein 1-mediated thermogenic capacity. Furthermore, mirabegron, a selective human β3-AR agonist, stimulated BAT lipolysis and thermogenesis, and both processes were lost after silencing β3-AR expression. This study highlights that β3-ARs in human brown/beige adipocytes are required to maintain multiple components of the lipolytic and thermogenic cellular machinery and that β3-AR agonists could be used to achieve metabolic benefit in humans.

Objective: Beige/brite adipose tissue displays morphological characteristics and beneficial metabolic traits of brown adipose tissue. Previously, we showed that TGF-β signaling regulates the browning of white adipose tissue. Here, we inquired whether TGF-β signals regulated presumptive beige progenitors in white fat and investigated the TGF-β regulated mechanisms involved in beige adipogenesis. Methods: We deleted TGF-β receptor 1 (TβRI) in adipose tissue (TβRIAdKO mice) and, using flow-cytometry based assays, identified and isolated presumptive beige progenitors located in the stromal vascular cells of white fat. These cells were molecularly characterized to examine beige/brown marker expression and to investigate TGF-β dependent mechanisms. Further, the cells were transplanted into athymic nude mice to examine their adipogenesis potential. Results: Deletion of TβRI promotes beige adipogenesis while reducing the detrimental effects of high fat diet feeding. Interaction of TGF-β signaling with the prostaglandin pathway regulated the appearance of beige adipocytes in white fat. Using flow cytometry techniques and stromal vascular fraction from white fat, we isolated presumptive beige stem/progenitor cells (iBSCs). Upon genetic or pharmacologic inhibition of TGF-β signaling, these cells express high levels of predominantly beige markers. Transplantation of TβRI-deficient stromal vascular cells or iBSCs into athymic nude mice followed by high fat diet feeding and stimulation of β-adrenergic signaling via CL316,243 injection or cold exposure promoted robust beige adipogenesis in vivo. Conclusions: TβRI signals target the prostaglandin network to regulate presumptive beige progenitors in white fat capable of developing into beige adipocytes with functional attributes. Controlled inhibition of TβRI signaling and concomitant PGE2 stimulation has the potential to promote beige adipogenesis and improve metabolism. Keywords: Beige/brite adipogenesis, Progenitors, Metabolism, Diabetes, Obesity, TGF-beta, Prostaglandin E2, Cyclooxygenase 2

Brown adipose tissue (BAT) in adult humans has been recently rediscovered and intensively investigated as a new potential therapeutic target for obesity and type 2 diabetes (T2D). However, reliable assessment of BAT mass in vivo represents a considerable challenge. The purpose of this investigation is to demonstrate for the first time that human BAT depots can be imaged with a translocator protein (TSPO)-specific positron emission tomography (PET) tracer [11C]PBR28 under thermoneutral conditions. In this retrospective analysis, we analyzed the images of three healthy volunteers who underwent PET/magnetic resonance (MR) imaging after injection of 14 m Ci of [11C]PBR28 at room temperature. Thirty-minute static PET images were reconstructed from the data obtained 60–90 min after the injection of the tracer. [11C]PBR28 uptake in the neck/supraclavicular regions was identified, which was parallel to the known distribution pattern of human BAT depots. These areas co-localized with the areas of hyperintensity and corresponded to fat on T1-weighted MR images. Standardized uptake value (SUV) was used to quantify [11C]PBR28 signal in BAT depots. The average (± SD) SUV(mean) and SUVmax for BAT depots was 2.13 (± 0.33) and 3.19 (± 0.34), respectively, while the average SUV(mean) for muscle and subcutaneous adipose tissue was 0.79 (± 0.1) and 0.18 (± 0.04), respectively. In this brief article, we provide the first evidence suggesting that [11C]PBR28, a widely available TSPO-specific PET tracer, can be used for imaging human BAT mass under thermoneutral conditions.

Using a number of cell biological and statistical methods we identify and characterize EIF5, TCF25 and BIN1 as markers for individual brown adipocyte subtypes in C57BL/6J mice., Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. Although increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Recently, UCP1 high and low expressing brown adipocytes were identified, but a developmental origin of these subtypes has not been studied. To obtain more insights into brown preadipocyte heterogeneity, we use single-cell RNA sequencing of the BAT stromal vascular fraction of C57/BL6 mice and characterize brown preadipocyte and adipocyte clonal cell lines. Statistical analysis of gene expression profiles from brown preadipocyte and adipocyte clones identify markers distinguishing brown adipocyte subtypes. We confirm the presence of distinct brown adipocyte populations in vivo using the markers EIF5, TCF25, and BIN1. We also demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that BIN1 marks dormant brown adipocytes. The existence of multiple brown adipocyte subtypes suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct functions in thermogenesis and the regulation of whole body energy homeostasis.

Persons with HIV are at increased risk for adipose dysfunction, which could mediate metabolic complications such as cardiovascular disease, fatty liver disease, and diabetes. We have previously reported reduced browning and beiging capacity of the subcutaneous adipose depot in HIV.We sought to evaluate how HIV-related parameters are related to the expression of brown and beige fat genes in the abdominal subcutaneous adipose tissue.Eighteen persons with HIV underwent punch biopsy of abdominal subcutaneous fat to determine mRNA expression of adipose-related genes using quantitative reverse transcriptase-polymerase chain reaction.Duration of antiretroviral therapy use, particularly related to protease inhibitor use, was significantly related to reduced expression of multiple brown and beige fat genes (including UCP1, PGC1α, PRDM16 and others, all P ≤ 0.04) in the abdominal subcutaneous fat. In addition, duration of HIV and CD4 T-cell count were significantly correlated with reduced expression of multiple brown and beige fat genes in the abdominal subcutaneous fat (PGC1α, P2XR5, TMEM26, CD137, all P ≤ 0.05 for duration of HIV; and PGC1α, ZIC1, PRDM16, PAT2, P2RX5, TMEM26, CD137, all P ≤ 0.04). In contrast, HIV viral load did not correlate with any brown or beige fat genes.Key HIV-related parameters reflective of nonacute infection (increased duration of HIV and duration of antiretroviral therapy use) or relatively reduced immunologic function (lower CD4 count) were linked to reduced expression of brown and beige fat gene in the abdominal subcutaneous adipose depot.NCT01098045.

Human obesity is an increasing burden worldwide. Lipids stored as metabolic fuel in white adipose tissue (WAT) can be rapidly mobilized through lipolysis. In rodent adipocytes, activation of the β3-adrenergic receptors (β3-ARs) leads to lipolysis. The physiological role of the β3-AR in human adipocytes remains controversial due to its reported low expression and previous access only to partial β3-AR agonists. Mirabegron, an FDA-approved drug for overreactive bladder, is a highly-selective human β3-AR agonist. In clinical trials, mirabegron treatment led to activation of human brown adipose tissue (BAT) thermogenesis and increased plasma NEFA’s. Therefore, we investigated if mirabegron directly mediates lipolysis in human white adipocytes. We found similar gene expression of the three β-ARs in human immortalized and primary white adipocytes, with β2-AR being the most expressed, followed by β1-AR and β3-AR. Next, to assess the functionally of the β3-AR in human adipocytes, we established dose-response curves and found that mirabegron increased the release of glycerol, a lipolytic product, in the picomolar range. To identify the molecular mechanism controlling mirabegron-induced lipolysis, we assessed the regulation of key downstream lipolytic proteins. Mirabegron mediated lipolysis by rapidly phosphorylating hormone sensitive lipase (HSL), as well as the mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Moreover, acute inhibition of either the PKA or MAPK/ERK pathways decreased lipolysis. In summary, we identify a major role played by the β3-AR in the regulation of human WAT lipolysis. Mirabegron acts as a potent pro-lipolytic effector, signaling through the canonical adrenergic receptor pathway as well as the MAPK/ERK pathway. Due to the comparatively minimal effects previously reported of mirabegron on the cardiovascular system, mirabegron may be the optimal choice for targeted lipid mobilization in human WAT. Disclosure C. Cero: None. J.W. Johnson: None. A. O’Mara: None. J.D. Linderman: None. A.M. Cypess: None. Funding National Institutes of Health

Objective This study aimed to quantify and compare the amount, activity, and anatomical distribution of cold-activated brown adipose tissue (BAT) in healthy, young, lean women and men. Methods BAT volume and 18 F-fluorodeoxyglucose uptake were measured by positron emission tomography and computerized tomography in 12 women and 12 men (BMI 18.5-25 kg/m2 , aged 18-35 years) after 5 hours of exposure to their coldest temperature before overt shivering. Results Women had a lower detectable BAT volume than men (P = 0.03), but there was no difference after normalizing to body size. The mean BAT glucose uptake and relative distribution of BAT did not differ by sex. 18 F-fluorodeoxyglucose uptake consistent with BAT was observed in superficial dorsocervical adipose tissue of 6 of 12 women but only 1 of 12 men (P = 0.02). This potential BAT depot would pose fewer biopsy risks than other depots. Conclusions Despite differences in adiposity and total BAT volume, we found that healthy, lean, young women and men do not differ in the relative amount, glucose uptake, and distribution of BAT. Dorsocervical 18 F-fluorodeoxyglucose uptake was more prevalent in women and may be a remnant of interscapular BAT seen in human newborns. Future studies are needed to discern how BAT contributes to whole-body thermal physiology and body weight regulation in women and men.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT's ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT's thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT's ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT's thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Brown adipose tissue (BAT) activation after cold exposure and its subsequent detection via18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) is mediated through norepinephrine (NE) induced β3-adrenoceptor activation. Pheochromocytoma/paragangliomas (PPGLs), especially SDH, VHL, and FH germline variants (cluster 1 tumors), predominantly secrete NE and often has higher maximal standardized uptake values of tumors on18F-FDG PET/CT1. Therefore, 18F-FDG PET/CT scans of these NE-secreting PPGLs should also reveal higher BAT activity. Because BAT activation is also associated with cancer-induced cachexia and thus poor outcomes 2, BAT activation in PPGLs could also indicate aggressive tumor behavior. To study the relationship between BAT activation detected on 18F-FDG PET/CT and the clinical behavior of PPGLs, we performed a retrospective study of 343 patients with genetically characterized and histopathologically confirmed PPGLs who underwent 18F-FDG PET/CT imaging for disease surveillance between 2013 and 2017. After excluding patients with only post-operative PET scans and those with normal serum catecholamines and/or metanephrines, 205 patients were included in the final analysis. BAT activation was observed in 16 patients (7.8%; 28±17y; 10F/6M, 23.4±4.2 kg/m2). Hereditary PPGLs were present in 12/16 patients of which, 10 had cluster 1 variants (SDHx, n=7; HIF2A, n=2; VHL, n=1), 2 had cluster 2 variants (NF1, n=2), and 4 had sporadic PPGL. Elevated plasma biomarkers were as follows: epinephrine (n=0/16); metanephrine (n=2/16); NE (n=11/16); normetanephrine (n=16/16); and dopamine (n=8/16). A matched control group of 36 PPGL patients without detectable BAT activation (34±13y, 21F/15M,23.9±2.5 kg/m2, 12/36 SDHx, p=0.47) revealed a significantly lower mortality rate (n=2/36 vs n=4/16; p=0.043). All deaths in the BAT activation group were found in patients with SDHx variants compared to sporadic PPGL patients in the control group. The development of metastasis was similar in both groups (n=10/16, 62.5% vs. n=27/36, 75%; p=0.36). In conclusion, cluster 1 PPGLs are more likely to be associated with BAT activation. We show for the first time that particularly the combination of an SDHx mutation and BAT activation is associated with a higher mortality. Further investigation is necessary to establish the mechanistic connection between PPGLs, especially those with SDHx mutations, BAT activation, and poorer prognosis. References: 1-Martucci VL, Pacak K. Pheochromocytoma and paraganglioma: diagnosis, genetics, management, and treatment. Current problems in cancer. 2014;38(1):7-41. 2-Petruzzelli M, Schweiger M, Schreiber R, et al. A switch from white to brown fat increases energy expenditure in cancer-associated cachexia. Cell metabolism. 2014;20(3):433-447.

ObjectiveCold exposure increases energy expenditure (EE) and could have a role in combating obesity. To understand this potential, we determined the capacity for cold-induced thermogenesis (CIT), the EE increase above the basal metabolic rate at the individualized coldest tolerable temperature before overt shivering.DesignDuring a 13-day inpatient protocol, we quantitated the EE of 12 lean men and 9 men with obesity at various randomly ordered ambient temperatures in a room calorimeter. Subjects underwent brown fat imaging after exposure to their coldest tolerable temperature.ResultsCIT capacity was 300 ± 218 kcal/d (mean ± SD) or 17 ± 11% in lean men and 125 ± 146 kcal/d or 6 ± 7% in men with obesity (P = 0.01). The temperature below which EE increased, lower critical temperature (Tlc), was warmer in lean men than men with obesity (22.9 ± 1.2 vs 21.1 ± 1.7°C, P = 0.03), but both had similar skin temperature (Tskin) changes and coldest tolerable temperatures. Whereas lean subjects had higher brown fat activity, skeletal muscle activity increased synchronously with CIT beginning at the Tlc in both groups, indicating that muscle is recruited for CIT in parallel with brown fat, not sequentially after nonshivering thermogenesis is maximal.ConclusionsDespite greater insulation from fat, men with obesity had a narrower range of tolerable cool temperatures available for increasing EE and less capacity for CIT than lean men, likely as a result of greater basal heat production and similar perception to Tskin cooling. Further study of the reduced CIT capacity in men with obesity may inform treatment opportunities for obesity.

In addition to its role in adaptive thermogenesis, brown adipose tissue (BAT) may protect from weight gain, insulin resistance/diabetes, and metabolic syndrome. Prior studies have shown contradictory results regarding the influence of thyroid hormone (TH) levels on BAT volume and activity. The aim of this pilot study was to gain further insights regarding the effect of TH treatment on BAT function in adult humans by evaluating the BAT mass and activity prospectively in six patients, first in the hypothyroid and then in the thyrotoxic phase.The study subjects underwent 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) scanning after cold exposure to measure BAT mass and activity while undergoing treatment for differentiated thyroid cancer, first while hypothyroid following TH withdrawal at the time of the radioactive iodine treatment and then three to six months after starting TH suppressive treatment when they were iatrogenically thyrotoxic. Thermogenic and metabolic parameters were measured in both phases.All study subjects had detectable BAT under cold stimulation in both the hypothyroid and thyrotoxic state. The majority but not all (4/6) subjects showed an increase in detectable BAT volume and activity under cold stimulation between the hypothyroid and thyrotoxic phase (total BAT volume: 72.0 ± 21.0 vs. 87.7 ± 16.5 mL, p = 0.25; total BAT activity 158.1 ± 72.8 vs. 189.0 ± 55.5 SUV*g/mL, p = 0.34). Importantly, circulating triiodothyronine was a stronger predictor of energy expenditure changes compared with cold-induced BAT activity.Iatrogenic hypothyroidism lasting two to four weeks does not prevent cold-induced BAT activation, while the use of TH to induce thyrotoxicosis does not consistently increase cold-induced BAT activity. It remains to be determined which physiological factors besides TH play a role in regulating BAT function.

Background: Type-2 diabetes mellitus (T2DM) accelerates cognitive aging and increases risk of Alzheimer's disease. Rodent models of T2DM show altered synaptic plasticity associated with reduced learning and memory. Humans with T2DM also show cognitive deficits, including reduced learning and memory, but the relationship of these impairments to the efficacy of neuroplastic mechanisms has never been assessed. Objective: Our primary objective was to compare mechanisms of cortical plasticity in humans with and without T2DM. Our secondary objective was to relate plasticity measures to standard measures of cognition. Methods: A prospective cross-sectional cohort study was conducted on 21 adults with T2DM and 15 demographically similar non-diabetic controls. Long-term potentiation-like plasticity was assessed in primary motor cortex by comparing the amplitude of motor evoked potentials (MEPs) from single-pulse transcranial magnetic stimulation before and after intermittent theta-burst stimulation (iTBS). Plasticity measures were compared between groups and related to neuropsychological scores. Results: In T2DM, iTBS-induced modulation of MEPs was significantly less than controls, even after controlling for potential confounds. Furthermore, in T2DM, modulation of MEPs 10-min post-iTBS was significantly correlated with Rey Auditory Verbal Learning Task (RAVLT) performance. Conclusion: Humans with T2DM show abnormal cortico-motor plasticity that is correlated with reduced verbal learning. Since iTBS after-effects and the RAVLT are both NMDA receptor-dependent measures, their relationship in T2DM may reflect brain-wide alterations in the efficacy of NMDA receptors. These findings offer novel mechanistic insights into the brain consequences of T2DM and provide a reliable means to monitor brain health and evaluate the efficacy of clinical interventions.

"Beige" adipocytes reside in white adipose tissue (WAT) and dissipate energy as heat. Several studies have shown that cold temperature can activate pro-opiomelanocortin-expressing (POMC) neurons and increase sympathetic neuronal tone to regulate WAT beiging. WAT, however, is traditionally known to be sparsely innervated. Details regarding the neuronal innervation and, more importantly, the propagation of the signal within the population of "beige" adipocytes are sparse. Here, we demonstrate that beige adipocytes display an increased cell-to-cell coupling via connexin 43 (Cx43) gap junction channels. Blocking of Cx43 channels by 18α-glycyrrhetinic acid decreases POMC-activation-induced adipose tissue beiging. Adipocyte-specific deletion of Cx43 reduces WAT beiging to a level similar to that observed in denervated fat pads. In contrast, overexpression of Cx43 is sufficient to promote beiging even with mild cold stimuli. These data reveal the importance of cell-to-cell communication, effective in cold-induced WAT beiging, for the propagation of limited neuronal inputs in adipose tissue.

Evidence suggests abnormal bioenergetic status throughout the body in psychotic disorders. The present study examined predictors of elevated body mass index (BMI) across diagnostic categories of schizophrenia, schizoaffective and bipolar disorders. In a cross-sectional study, we studied demographic and clinical risk factors for overweight and obesity in a well-characterized sample of 262 inpatients and outpatients with schizophrenia (n=59), schizoaffective disorder (n=81) and bipolar I disorder (n=122). Across the three diagnostic categories, the prevalence of overweight (29.4%) and obesity (33.2%) combined was 62.6% (164/262). Logistic regression analyses, adjusted for age, sex and ethnicity, showed that schizoaffective disorder, lifetime major depressive episode, presence of prior suicide attempt, and more than 5 lifetime hospitalizations were significantly associated with BMI≥25. Patients with schizophrenia had significantly lower risk for overweight and obesity. Overall, we found that affective components of illness were associated with elevated BMI in our cross-diagnostic sample. Our results show that patients with schizoaffective disorder have a greater risk for obesity. Identifying predictors of elevated BMI in patients with psychotic and mood disorders will help prevent obesity and related cardiovascular and cerebral complications. Future studies are needed to elucidate the mechanistic nature of the relationship between obesity and psychiatric illness.

Patients with psychotic disorders are at high risk for type 2 diabetes mellitus, and there is increasing evidence that patients display glucose metabolism abnormalities before significant antipsychotic medication exposure. In the present study, we examined insulin action by quantifying insulin sensitivity in first-episode psychosis (FEP) patients and unaffected siblings, compared to healthy individuals, using a physiological-based model and comprehensive assessment battery. Twenty-two unaffected siblings, 18 FEP patients, and 15 healthy unrelated controls were evaluated using a 2-h oral glucose tolerance test (OGTT), with 7 samples of plasma glucose and serum insulin concentration measurements. Insulin sensitivity was quantified using the oral minimal model method. Lipid, leptin, free fatty acids, and inflammatory marker levels were also measured. Anthropometric, nutrient, and activity assessments were conducted; total body composition and fat distribution were determined using whole-body dual-energy X-ray absorptiometry. Insulin sensitivity significantly differed among groups (F = 6.01 and 0.004), with patients and siblings showing lower insulin sensitivity, compared to controls (P = 0.006 and 0.002, respectively). Body mass index, visceral adipose tissue area (cm2), lipids, leptin, free fatty acids, inflammatory markers, and activity ratings were not significantly different among groups. There was a significant difference in nutrient intake with lower total kilocalories/kilogram body weight in patients, compared to siblings and controls. Overall, the findings suggest that familial abnormal glucose metabolism or a primary insulin signaling pathway abnormality is related to risk for psychosis, independent of disease expression and treatment effects. Future studies should examine underlying biological mechanisms of insulin signaling abnormalities in psychotic disorders.