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Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

The β3 -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [3 H]-2-deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β3 -adrenoceptors as they were largely absent in adipocytes derived from β3 -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β3 -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β3 -adrenoceptor.

The β3‐adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity‐related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β‐adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron‐mediated effects were examined on cyclic AMP, UCP1 mRNA, [3H]‐2‐deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1‐mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β3‐adrenoceptors as they were largely absent in adipocytes derived from β3‐adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β3‐adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β3‐adrenoceptor.

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.

Osteocytes reside within bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been difficult to study in vitro because they are difficult to extract and purify, and do not retain their phenotype in standard culture conditions. However, new techniques for this purpose are emerging. This chapter will describe three methods we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic-lineage depletion, (2) differentiating cultured osteoblasts (or osteoblast cell lines) until they reach a stage of osteocytic gene expression, and (3) using the Ocy454 osteocyte-like cell line.

Osteocytes reside within bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been difficult to study in vitro because they are difficult to extract and purify, and do not retain their phenotype in standard culture conditions. However, new techniques for this purpose are emerging. This chapter will describe three methods we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic-lineage depletion, (2) differentiating cultured osteoblasts (or osteoblast cell lines) until they reach a stage of osteocytic gene expression, and (3) using the Ocy454 osteocyte-like cell line.

A vital role of adrenoceptors in metabolism and energy balance has been well documented in the heart, skeletal muscle, and adipose tissue. It has been only recently demonstrated, however, that activation of the mechanistic target of rapamycin (mTOR) makes a significant contribution to various metabolic and physiological responses to adrenoceptor agonists. mTOR exists as two distinct complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) and has been shown to play a critical role in protein synthesis, cell proliferation, hypertrophy, mitochondrial function, and glucose uptake. This review will describe the physiological significance of mTORC1 and 2 as a novel paradigm of adrenoceptor signalling in the heart, skeletal muscle, and adipose tissue. Understanding the detailed signalling cascades of adrenoceptors and how they regulate physiological responses is important for identifying new therapeutic targets and identifying novel therapeutic interventions. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β3-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β3-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β3-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β3-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β3-adrenoceptor expression and provides an improved window for detection of β3-adrenoceptor responses.

To define their gene expression and function, osteocytes are commonly isolated and purified by fluorescence-activated cell sorting (FACS) from mice expressing GFP directed by the dentin matrix protein 1 (Dmp1) promoter (DMP1-GFP). These cells express mRNA for osteocyte genes, including sclerostin (Sost) and Dmp1, and genes associated with the osteoclast phenotype: Dcstamp, Oscar, Cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAP/Acp5) and calcitonin receptor (Calcr). This suggests either that osteoclasts and osteocytes share genes and functions or that DMP1-GFP(+) preparations contain haematopoietic osteoclasts. To resolve this we stained DMP1-GFP cells for haematopoietic lineage (Lin) surface markers (CD2, CD3e, CD4, CD45, CD5, CD8, CD11b, B220, Gr1, Ter119) and CD31. Lin(-)CD31(-) (Lin(-)) and Lin(+)CD31(+) (Lin(+)) populations were analysed for GFP, and the four resulting populations assessed by quantitative real-time PCR. Lin(-)GFP(+) cells expressed mRNAs for Sost, Dmp1, and Mepe, confirming their osteocyte identity. Dcstamp and Oscar mRNAs were restricted to haematopoietic (Lin(+)) cells, but Calcr, Ctsk and Acp5 were readily detected in purified osteocytes (Lin(-)GFP(+)). The capacity of these purified osteocytes to support osteoclastogenesis was assessed: no TRAP+ cells with >2 nuclei were formed when purified osteocytes were cultured with bone marrow macrophages and stimulated with 1,25-dihydroxyvitamin-D3/prostaglandin E2. Lin(-)GFP(+) osteocytes also expressed lower levels of Tnfsf11 (RANKL) mRNA than the osteoblast-enriched population (Lin(-)GFP(-)). This demonstrates the importance of haematopoietic depletion in generating highly purified osteocytes and shows that osteocytes express Acp5, Ctsk and Calcr, but not other osteoclast markers, and do not fully support osteoclast formation in vitro.

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α

Although it is well established that exogenous androgens have anabolic effects on skeletal muscle mass in humans and mice, data from muscle-specific androgen receptor (AR) knockout (ARKO) mice indicate that myocytic expression of the AR is dispensable for hind-limb muscle mass accrual in males. To identify possible indirect actions of androgens via the AR in neurons to regulate muscle, we generated neuron-ARKO mice in which the dominant DNA binding-dependent actions of the AR are deleted in neurons of the cortex, forebrain, hypothalamus, and olfactory bulb. Serum testosterone and luteinizing hormone levels were elevated twofold in neuron-ARKO males compared with wild-type littermates due to disruption of negative feedback to the hypothalamic-pituitary-gonadal axis. Despite this increase in serum testosterone levels, which was expected to increase muscle mass, the mass of the mixed-fiber gastrocnemius (Gast) and the fast-twitch fiber extensor digitorum longus hind-limb muscles was decreased by 10% in neuron-ARKOs at 12 weeks of age, whereas muscle strength and fatigue of the Gast were unaffected. The mass of the soleus muscle, however, which consists of a high proportion of slow-twitch fibers, was unaffected in neuron-ARKOs, demonstrating a stimulatory action of androgens via the AR in neurons to increase the mass of fast-twitch hind-limb muscles. Furthermore, neuron-ARKOs displayed reductions in voluntary and involuntary physical activity by up to 60%. These data provide evidence for a role of androgens via the AR in neurons to positively regulate fast-twitch hind-limb muscle mass and physical activity in male mice.

Sclerostin, a secreted protein encoded by the SOST gene, has been identified as a key regulator of bone formation by studies in human and mouse genetics. Expression of this protein in osteocytes has been shown to be regulated by mechanical forces, and this has been shown to be critical for the bone formation response to load. Osteocytic expression of sclerostin is also regulated by systemic hormones that are known to influence the skeleton including parathyroid hormone and calcitonin. Circulating levels of sclerostin appear to be influenced by circulating sex steroid levels. Paracrine and autocrine factors expressed by the cells within bone (osteoblasts, osteoclasts and osteocytes), including some members of the family of cytokines that signal through gp130 (oncostatin M, cardiotrophin-1 and leukemia inhibitory factor) as well as prostaglandin E2, rapidly regulate osteocytic sclerostin expression, pointing to new paracrine networks within the bone matrix. In addition, regulation of sclerostin by osteoblastic transcription factors, such as osterix and zinc finger protein 467, has been identified. Finally, changes in sclerostin expression due to changes in osteoblast differentiation have been reported in response to inhibitors of ephrin signaling and in response to the inflammatory mediators tumor necrosis factor and TWEAK. This review will discuss the evidence for each of these influences and what they might mean for bone physiology.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5 h after administration of a single injection of PTH, sCT or PTH + sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

We have previously shown that co-administration of the transient osteoclast inhibitor, salmon calcitonin (sCT), blunts the anabolic effect of parathyroid hormone (PTH) in young rats and increases osteocytic expression of the bone formation inhibitor sclerostin (Sost). To determine whether this also occurs in adult animals, we co-administered sCT with PTH to 6-month-old sham-operated (SHAM) and ovariectomised (OVX) rats. While sCT reduced the stimulatory effect of PTH on serum amino-terminal propeptide of type 1 procollagen levels, in contrast to its influence in young rats, sCT did not reduce the anabolic effect of PTH on femoral bone mineral density, tibial trabecular bone volume or bone formation rate in 6-month-old SHAM or OVX rats. Quantitative real-time PCR analysis of femoral metaphyses collected 1 and 4 h after a single PTH injection confirmed a significant increase in mRNA levels for interleukin 6 (Il6) and ephrinB2 (EfnB2), and a significant reduction in Sost and dentin matrix protein-1 (Dmp1) in response to PTH. However, in contrast to observations in young rats, these effects were not modified by co-administration of sCT, nor did sCT significantly modify Sost, Dmp1, or matrix extracellular phosphoglycoprotein (Mepe) mRNA levels. Furthermore, while CT receptor (CTR) mRNA (Calcr) was readily detected in GFP+ osteocytes isolated from young (3-week-old) DMP1–GFP mice, Calcr levels in osteocytes declined as mice aged, reaching levels that were undetectable in long bone at 49 weeks of age. These data indicate that osteocyte-mediated responses to CT are most likely to be of physiological relevance in young rodents.