Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-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.

A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Origin and Function of Stress-Induced IL-6 in Murine Models.

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.

GDF15 Is an Inflammation-Induced Central Mediator of Tissue Tolerance.

Growth and differentiation factor 15 (GDF15) is an inflammation-associated hormone with poorly defined biology. Here, we investigated the role of GDF15 in bacterial and viral infections. We found that inflammation induced GDF15, and that GDF15 was necessary for surviving both bacterial and viral infections, as well as sepsis. The protective effects of GDF15 were largely independent of pathogen control or the magnitude of inflammatory response, suggesting a role in disease tolerance. Indeed, we found that GDF15 was required for hepatic sympathetic outflow and triglyceride metabolism. Failure to defend the lower limit of plasma triglyceride levels was associated with impaired cardiac function and maintenance of body temperature, effects that could be rescued by exogenous administration of lipids. Together, we show that GDF15 coordinates tolerance to inflammatory damage through regulation of triglyceride metabolism.

The Molecular Basis of G Protein-Coupled Receptor Activation.

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and ßγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells.

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

Crosstalk between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity.

Adrenergic stimulation promotes lipid mobilization and oxidation in brown and beige adipocytes, where the harnessed energy is dissipated as heat in a process known as adaptive thermogenesis. The signaling cascades and energy-dissipating pathways that facilitate thermogenesis have been extensively described, yet little is known about the counterbalancing negative regulatory mechanisms. Here, we identify a two-pore-domain potassium channel, KCNK3, as a built-in rheostat negatively regulating thermogenesis. Kcnk3 is transcriptionally wired into the thermogenic program by PRDM16, a master regulator of thermogenesis. KCNK3 antagonizes norepinephrine-induced membrane depolarization by promoting potassium efflux in brown adipocytes. This limits calcium influx through voltage-dependent calcium channels and dampens adrenergic signaling, thereby attenuating lipolysis and thermogenic respiration. Adipose-specific Kcnk3 knockout mice display increased energy expenditure and are resistant to hypothermia and obesity. These findings uncover a critical K+-Ca2+-adrenergic signaling axis that acts to dampen thermogenesis, maintain tissue homeostasis, and reveal an electrophysiological regulatory mechanism of adipocyte function.

Structural and Functional Analysis of a ß2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Cryo-EM structure of the ß3-adrenergic receptor reveals the molecular basis of subtype selectivity.

The ß3-adrenergic receptor (ß3AR) is predominantly expressed in adipose tissue and urinary bladder and has emerged as an attractive drug target for the treatment of type 2 diabetes, obesity, and overactive bladder (OAB). Here, we report the cryogenic electron microscopy structure of the ß3AR-Gs signaling complex with the selective agonist mirabegron, a first-in-class drug for OAB. Comparison of this structure with the previously reported ß1AR and ß2AR structures reveals a receptor activation mechanism upon mirabegron binding to the orthosteric site. Notably, the narrower exosite in ß3AR creates a perpendicular pocket for mirabegron. Mutational analyses suggest that a combination of both the exosite shape and the amino-acid-residue substitutions defines the drug selectivity of the ßAR agonists. Our findings provide a molecular basis for ßAR subtype selectivity, allowing the design of more-selective agents with fewer adverse effects.

Cold-responsive adipocyte progenitors couple adrenergic signaling to immune cell activation to promote beige adipocyte accrual.

The full array of cold-responsive cell types within white adipose tissue that drive thermogenic beige adipocyte biogenesis remains undefined. We demonstrate that acute cold challenge elicits striking transcriptomic changes specifically within DPP4+ PDGFRß+ adipocyte precursor cells, including a ß-adrenergic receptor CREB-mediated induction in the expression of the prothermogenic cytokine, Il33 Doxycycline-inducible deletion of Il33 in PDGFRß+ cells at the onset of cold exposure attenuates ILC2 accumulation and beige adipocyte accrual. These studies highlight the multifaceted roles for adipocyte progenitors and the ability of select mesenchymal subpopulations to relay neuronal signals to tissue-resident immune cells in order to regulate tissue plasticity.

Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal Lung.

Young children are more susceptible to developing allergic asthma than adults. As neural innervation of the peripheral tissue continues to develop after birth, neurons may modulate tissue inflammation in an age-related manner. Here we showed that sympathetic nerves underwent a dopaminergic-to-adrenergic transition during post-natal development of the lung in mice and humans. Dopamine signaled through a specific dopamine receptor (DRD4) to promote T helper 2 (Th2) cell differentiation. The dopamine-DRD4 pathway acted synergistically with the cytokine IL-4 by upregulating IL-2-STAT5 signaling and reducing inhibitory histone trimethylation at Th2 gene loci. In murine models of allergen exposure, the dopamine-DRD4 pathway augmented Th2 inflammation in the lungs of young mice. However, this pathway operated marginally after sympathetic nerves became adrenergic in the adult lung. Taken together, the communication between dopaminergic nerves and CD4+ T cells provides an age-related mechanism underlying the susceptibility to allergic inflammation in the early lung.

Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound ß1-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Sympathetic Neuronal Activation Triggers Myeloid Progenitor Proliferation and Differentiation.

There is a growing body of research on the neural control of immunity and inflammation. However, it is not known whether the nervous system can regulate the production of inflammatory myeloid cells from hematopoietic progenitor cells in disease conditions. Myeloid cell numbers in diabetic patients were strongly correlated with plasma concentrations of norepinephrine, suggesting the role of sympathetic neuronal activation in myeloid cell production. The spleens of diabetic patients and mice contained higher numbers of tyrosine hydroxylase (TH)-expressing leukocytes that produced catecholamines. Granulocyte macrophage progenitors (GMPs) expressed the ß2 adrenergic receptor, a target of catecholamines. Ablation of splenic sympathetic neuronal signaling using surgical, chemical, and genetic approaches diminished GMP proliferation and myeloid cell development. Finally, mice lacking TH-producing leukocytes had reduced GMP proliferation, resulting in diminished myelopoiesis. Taken together, our study demonstrates that catecholamines produced by leukocytes and sympathetic nerve termini promote GMP proliferation and myeloid cell development.

Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

Internalized ß2-Adrenergic Receptors Oppose PLC-Dependent Hypertrophic Signaling.

BACKGROUND: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR (ß1-adrenergic receptor) and ß2-ARs (ß2-adrenergic receptor) are the 2 major subtypes of ß-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1-ARs drives detrimental cardiac remodeling while ß2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2-ARs remain unclear. METHODS: ß2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of ß2-AR signaling mechanisms in the regulation of cardiac hypertrophy. RESULTS: Here, we show that ß2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for ß2-AR-mediated phospholipase C inhibition requires internalization of ß2-AR, activation of Gi and Gßγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. CONCLUSIONS: This reveals a mechanism for ß2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.