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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

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.

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.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3., Cell, 184 (13), ISSN:0092-8674, ISSN:1097-4172

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.

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.

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.

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.

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.

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

Background Stress-associated conditions such as psychoemotional reactivity and depression have been paradoxically linked to either weight gain or weight loss. This bi-directional effect of stress is not understood at the functional level. Here we tested the hypothesis that pre-stress level of adaptive thermogenesis and brown adipose tissue (BAT) functions explain the vulnerability or resilience to stress-induced obesity. Methods We used wt and triple β1,β2,β3−Adrenergic Receptors knockout (β-less) mice exposed to a model of chronic subordination stress (CSS) at either room temperature (22 °C) or murine thermoneutrality (30 °C). A combined behavioral, physiological, molecular, and immunohistochemical analysis was conducted to determine stress-induced modulation of energy balance and BAT structure and function. Immortalized brown adipocytes were used for in vitro assays. Results Departing from our initial observation that βARs are dispensable for cold-induced BAT browning, we demonstrated that under physiological conditions promoting low adaptive thermogenesis and BAT activity (e.g. thermoneutrality or genetic deletion of the βARs), exposure to CSS acted as a stimulus for BAT activation and thermogenesis, resulting in resistance to diet-induced obesity despite the presence of hyperphagia. Conversely, in wt mice acclimatized to room temperature, and therefore characterized by sustained BAT function, exposure to CSS increased vulnerability to obesity. Exposure to CSS enhanced the sympathetic innervation of BAT in wt acclimatized to thermoneutrality and in β-less mice. Despite increased sympathetic innervation suggesting adrenergic-mediated browning, norepinephrine did not promote browning in βARs knockout brown adipocytes, which led us to identify an alternative sympathetic/brown adipocytes purinergic pathway in the BAT. This pathway is downregulated under conditions of low adaptive thermogenesis requirements, is induced by stress, and elicits activation of UCP1 in wt and β-less brown adipocytes. Importantly, this purinergic pathway is conserved in human BAT. Conclusion Our findings demonstrate that thermogenesis and BAT function are determinant of the resilience or vulnerability to stress-induced obesity. Our data support a model in which adrenergic and purinergic pathways exert complementary/synergistic functions in BAT, thus suggesting an alternative to βARs agonists for the activation of human BAT., Highlights • Adaptive thermogenesis/BAT functions determine the vulnerability to stress-induced obesity. • βARs are dispensable for stress and cold-induced BAT browning. • A sympathetic/brown adipocyte purinergic pathway is identified in mice and humans. • ATP increases UCP1 expression in brown adipocytes.

Background: Brown Adipose Tissue (BAT) in adult humans has become increasingly recognized to contribute to energy consumption and modulate metabolism. To date, BAT stimulation has predominately relied on cold exposure. However, long-term cold exposure is impractical as a treatment option for obesity and diabetes. Thus, we investigated if a chronic pharmacotherapeutic approach could stimulate BAT and contribute to metabolic health. Methods: In an ongoing study, 6 healthy female volunteers received 4 weeks of daily mirabegron (Myrbetriq, Astellas Pharma) 100 mg, a β3 adrenergic receptor (AR) agonist. Acute responses were determined by comparing effects before and then five hours after dosing. Chronic changes were assessed after 4 weeks of treatment. The primary endpoint was the change in BAT metabolic activity measured by 18F-FDG PET/CT. Secondary endpoints included resting energy expenditure (REE), insulin and glucose sensitivity, liver steatosis, and cardiovascular stimulation. Results: Acute effects of mirabegron included consistent increases in rate pressure product (RPP), REE, and serum non-esterified fatty acids (NEFA) (p Conclusions: Acute treatment with mirabegron in women successfully activates BAT thermogenesis. Preliminary data show chronic treatment resulted in a blunting of several responses, consistent with physiological attenuation of β-AR signaling. The increases in sleeping EE and improved insulin sensitivity suggest chronic mirabegron treatment may help alleviate metabolic disease. Disclosure A. O'Mara: None. A. Cypess: None. C. Cero: None. J.W. Johnson: None. J.D. Linderman: None. B. Leitner: None. L. Fletcher: None. R. Brychta: None. D. Kapuria: None. S. McGehee: None. Y. Rotman: None.

Obesity results from the accumulation of excess white adipose tissue, and its increasing rates worldwide pose a significant risk to overall health due to its multiple comorbidities, like type 2 diabetes. A novel treatment strategy is to increase energy expenditure through activation of brown and beige adipocytes, which can use uncoupling protein 1 (UCP1) for thermogenesis. In addition to having different developmental lineages, brown and beige adipocytes differ functionally regarding the additional mechanisms by which they generate heat. Most human fat depots are not easily accessible, so little is known about their developmental and functional characteristics. In this study, we collected adipose tissue from 11 patients (age 16-84, 4 female/7 male) who had undergone autopsy. We sampled from 7 anatomically distinct fat depots: subcutaneous (sc), omental (om), retroperitoneal (rp), pericardial (pc), periadrenal (pa), paraspinal (ps), and supraclavicular (sv). We measured mRNA levels of lineage and functional genes associated with brown, beige, and white adipocytes. Energy storing white adipocytes were defined by high leptin expression, while energy expending brown and beige cells were defined by high UCP1; these were further distinguished as having a brown lineage using Zic1 and beige via Tbx1. Principal component analysis showed three different clusters of genes consistent with the brown, beige, and white lineages. Functionally, based on expression of leptin and UCP1 for white and brown fat, respectively, the sc and om were white (P In summary, human adipose tissue is not merely white or brown but instead is composed of many depots with distinct developmental lineages and functional capacities. These differences will be important when trying to use brown and beige adipocyte thermogenesis to treat obesity and diabetes. Disclosure J.W. Johnson: None. C. Cero: None. A. O'Mara: None. J.D. Linderman: None. A.S. Baskin: None. A. Cypess: None.

In rodent models, the β3-adrenergic receptor (AR) is responsible for thermogenesis in brown adipose tissue (BAT) and lipolysis in white adipose tissue (WAT). In contrast, the contribution of β3-AR in human adipocytes is controversial due to the low expression of the β3-AR in WAT and access only to partial β3-AR agonists. Mirabegron, an FDA-approved drug for overreactive bladder, is a selective human β3-AR agonist. In a recent clinical trial, mirabegron activated human BAT thermogenesis and WAT lipolysis. Therefore, we investigated the distribution and relative contribution of β1/2/3-AR in (i) whole white (hWAT) and brown (hBAT) adipose tissue from human autopsies and (ii) immortalized human white (hWA) and brown (hBA) adipocytes. Among the three β-ARs, β1-AR and β2-AR are similarly expressed in brown and white adipose tissue, whereas β3-AR has the highest expression in hBAT. In immortalized differentiated cells, hBA have a significantly higher expression of β3-AR compared to hWA. To assess the functionally of these receptors in adipocytes, dose-response curves of agonist-stimulated lipolysis were investigated in differentiated human and mouse adipocytes. We observed that stimulation of any one of the three β-ARs in human adipocytes induces a significant increase in glycerol release, a lipolytic byproduct. However, the effective dose of mirabegron to activate β3-AR-mediated lipolysis in white and brown adipocytes via mirabegron was significantly lower (hWA: EC50 2.55E-09; hBA: EC50 6.31E-09) compared to specific activators of β1-AR or β2-AR. In summary, we identify a major role played by the β3-AR in the regulation of human BAT thermogenesis and WAT lipolysis, both in vivo and in vitro. Due to the comparatively minimal effects of mirabegron treatment on the cardiovascular system, selective β3-AR agonists may be the optimal choice for targeted stimulation of human WAT and BAT. Disclosure C. Cero: None. A. O'Mara: None. J.W. Johnson: None. A.S. Baskin: None. J.D. Linderman: None. A. Cypess: None.

TLQP-21 is a multifunctional neuropeptide and a promising new medicinal target for cardiometabolic and neurological diseases. However, to date its clearance kinetics and plasma stability have not been studied. The presence of four arginine residues led us to hypothesize that its half-life is relatively short. Conversely, its biological activities led us to hypothesize that the peptide is still taken up by adipose tissues effectively. [(125)I]TLQP-21 was i.v. administered in rats followed by chasing the plasma radioactivity and assessing tissue uptake. Plasma stability was measured using LC-MS. In vivo lipolysis was assessed by the palmitate rate of appearance. Results: A small single i. v. dose of [(125)I]TLQP-21 had a terminal half-life of 110 minutes with a terminal clearance rate constant, k(t), of 0.0063/min, and an initial half-life of 0.97 minutes with an initial clearance rate constant, k(i), of 0.71/min. The total net uptake by adipose tissue accounts for 4.4% of the entire dose equivalent while the liver, pancreas and adrenal gland showed higher uptake. Uptake by the brain was negligible, suggesting that i.v.-injected peptide does not cross the blood-brain-barrier. TLQP-21 sustained isoproterenol-stimulated lipolysis in vivo. Finally, TLQP-21 was rapidly degraded producing several N-terminal and central sequence fragments after 10 and 60 minutes in plasma in vitro. This study investigated the clearance and stability of TLQP-21 peptide for the first time. While its pro-lipolytic effect supports and extends previous findings, its short half-life and sequential cleavage in the plasma suggest strategies for chemical modifications in order to enhance its stability and therapeutic efficacy.

β3-adrenergic receptor (AR) agonists are approved to treat only overactive bladder. However, rodent studies suggest that these drugs could have other beneficial effects on human metabolism. We performed tissue receptor profiling and showed that the human β3-AR mRNA is also highly expressed in gallbladder and brown adipose tissue (BAT). We next studied the clinical implications of this distribution in 12 healthy men given one-time randomized doses of placebo, the approved dose of 50 mg, and 200 mg of the β3-AR agonist mirabegron. There was a more-than-dose-proportional increase in BAT metabolic activity as measured by [18F]-2-fluoro-D-2-deoxy-d-glucose positron emission tomography/computed tomography (medians 0.0 vs. 18.2 vs. 305.6 mL ⋅ mean standardized uptake value [SUVmean] ⋅ g/mL). Only the 200-mg dose elevated both nonesterified fatty acids (68%) and resting energy expenditure (5.8%). Previously undescribed increases in gallbladder size (35%) and reductions in conjugated bile acids were also discovered. Therefore, besides urinary bladder relaxation, the human β3-AR contributes to white adipose tissue lipolysis, BAT thermogenesis, gallbladder relaxation, and bile acid metabolism. This physiology should be considered in the development of more selective β3-AR agonists to treat obesity-related complications.

SUMMARY Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs., In Brief GPCRs and neuropeptide ligands are under intense evolutionary pressure and are major pharmacological targets. Sahu et al. identify a cluster of mutations within the C3aR1 receptor and the TLQP-21 peptide in the Murinae subfamily of rodents, resulting in enhanced binding affinity and potency, leading to potentiation of adrenergic-receptor-induced lipolysis., Graphical Abstract

Objectives Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. Methods We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, β1, β2, β3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. Results We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances β-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving β-adrenergic and C3a receptor activation without associated adverse metabolic effects. Conclusions In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules., Graphical abstract, Highlights • TLQP-21/C3aR1 does not possess lipolytic properties per se. • TLQP-21 enhances β-AR-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of HSL. • TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. • TLQP-21 treatment decreases body weight and fat mass in DIO mice by a mechanism involving β-AR and C3aR activation. • TLQP-21 anti-obesity effect is not associated with adverse metabolic effects.

Insulin secretion control is critical for glucose homeostasis. Paracrine and autocrine molecules secreted by cells of the islet of Langerhans, as well as by intramural and autonomic neurons, control the release of different hormones that modulate insulin secretion. In pancreatic islets, the abundant presence of the granin protein VGF (nonacronymic; unrelated to VEGF) suggests that some of its proteolytically derived peptides could modulate hormone release. Thus, in the present study, we screened several VGF-derived peptides for their ability to induce insulin secretion, and we identified the VGF C-terminal peptide TLQP-62 as the most effective fragment. TLQP-62 induced a potent increase in basal insulin secretion as well as in glucose-stimulated insulin secretion in several insulinoma cell lines. We found that this peptide stimulated insulin release via increased intracellular calcium mobilization and fast expression of the insulin 1 gene. Moreover, the peripheral injection of TLQP-62 in mice improved glucose tolerance. Together, the present findings suggest that TLQP-62, acting as an endocrine, paracrine, or autocrine factor, can be considered a new, strong insulinotropic peptide that can be targeted for innovative antidiabetic drug discovery programs.

TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled-receptor (GPCR) Complement-3a-Receptor1 (C3aR1). So far, the mechanisms of TLQP-21-induced receptor activation remained unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation, upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C-terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Interestingly, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions.

Stress and hypercaloric food are recognized risk factors for obesity, Metabolic Syndrome (MetS) and Type 2 Diabetes (T2D). Given the complexity of these metabolic processes and the unavailability of animal models, there is poor understanding of their underlying mechanisms. We established a model of chronic psychosocial stress in which subordinate mice are vulnerable to weight gain while dominant mice are resilient. Subordinate mice fed a standard diet showed marked hyperphagia, high leptin, low adiponectin, and dyslipidemia. Despite these molecular signatures of MetS and T2D, subordinate mice fed a standard diet were still euglycemic. We hypothesized that stress predisposes subordinate mice to develop T2D when synergizing with other risk factors. High fat diet aggravated dyslipidemia and the MetS thus causing a pre-diabetes-like state in subordinate mice. Contrary to subordinates, dominant mice were fully protected from stress-induced metabolic disorders when fed both a standard- and a high fat-diet. Dominant mice showed a hyperphagic response that was similar to subordinate but, unlike subordinates, showed a significant increase in VO2, VCO2, and respiratory exchange ratio when compared to control mice. Overall, we demonstrated a robust stress- and social status-dependent effect on the development of MetS and T2D and provided insights on the physiological mechanisms. Our results are reminiscent of the effect of the individual socioeconomic status on human health and provide an animal model to study the underlying molecular mechanisms.

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM–linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM–dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes., Author Summary REM and NREM sleep are two distinct stages of the sleeping brain that are involved in the modulation of metabolic, physiological, and cognitive processes. Clinical evidence suggests that epigenetic mechanisms, such as genomic imprinting, play a role in sleep. Here we show that REM and NREM brain states are differentially modulated by the maternally imprinted gene Gnas. In particular, a mutation resulting in loss of imprinted expression of Gnas enhances NREM–dependent physiologic and cognitive functions while repressing REM and REM-linked functions. This is the first experimental demonstration of a specific effect of genomic imprinting on sleep states and their associated effects on cognition.

The impact of stress is widely recognized in the etiology of multiple disorders. In particular, psychological stress may increase the risk of cardiovascular, metabolic, immune, and mood disorders. Several genes are considered potential candidates to account for the deleterious consequences of stress and recent data point to role of Vgf. VGF mRNA is abundantly expressed in the hypothalamus, where it has been involved in metabolism and energy homeostasis; more recently a link between VGF-derived peptides and mood disorders has been highlighted. The following experiments were performed to address the contribution of the VGF-system to stress induced changes in mice: the distribution of VGF immuno-reactivity in hypothalamic nuclei and its modulation by social stress; the role of VGF-derived peptide TLQP-21 in plasma catecholamine release induced by acute restraint stress (RS); the efficacy of chronic TLQP-21 in a mouse model of chronic subordination stress (CSS). VGF fibers were found in high density in arcuate, dorsomedial, and suprachiasmatic and, at lower density, in lateral, paraventricular, and ventromedial hypothalamic nuclei. Central administration of either 2 or 4 mM TLQP-21 acutely altered the biphasic serum epinephrine release and decreased norepinephrine serum levels in response to RS. Finally, 28-day of 40 μg/day TLQP-21 treatment increased CSS-induced social avoidance of an unfamiliar conspecific. Overall these data support a role for TLQP-21 in stress responses providing a promising starting point to further elucidate its role as a player in stress-related human pathologies.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptormediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesityassociated metabolic complications. © The Authors Journal compilation © 2012 Biochemical Society.