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Diet-induced obesity (DIO) reduces fatty acid oxidation in skeletal muscle and decreases circulating levels of adiponectin. Endocannabinoid signaling is overactive in obesity, with some effects abated by antagonism of cannabinoid receptor 1 (CB

BACKGROUND AND PURPOSE The physiological role of vascular β3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac β3 -adrenoceptors are functionally effective after down-regulation of β1 /β2 -adrenoceptors. The functional interaction between the β3 -adrenoceptor and other β-adrenoceptor subtypes in rat striated muscle arteries was investigated. EXPERIMENTAL APPROACH Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of β3 -adrenoceptor agonists and antagonists and other β-adrenoceptor ligands were measured using pressure myography. KEY RESULTS All three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The β3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β3 -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β1/2 -adrenoceptor antagonists nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The β3 -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β3 -adrenoceptor agonists (in the presence of β1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. CONCLUSION AND IMPLICATIONS All three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β3 -adrenoceptor mediated vasodilation was only evident after blockade of β1/2 -adrenoceptors. This suggests constitutive β1/2 -adrenoceptor activity inhibits β3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

The type 2 diabetes epidemic makes it important to find insulin-independent ways to improve glucose homeostasis. This study examines the mechanisms activated by a dual β2-/β3-adrenoceptor agonist, BRL37344, to increase glucose uptake in skeletal muscle and its effects on glucose homeostasis in vivo. We measured the effect of BRL37344 on glucose uptake, glucose transporter 4 (GLUT4) translocation, cAMP levels, β2-adrenoceptor desensitization, β-arrestin recruitment, Akt, AMPK, and mammalian target of rapamycin (mTOR) phosphorylation using L6 skeletal muscle cells as a model. We further tested the ability of BRL37344 to modulate skeletal muscle glucose metabolism in animal models (glucose tolerance tests and in vivo and ex vivo skeletal muscle glucose uptake). In L6 cells, BRL37344 increased GLUT4 translocation and glucose uptake only by activation of β2-adrenoceptors, with a similar potency and efficacy to that of the nonselective β-adrenoceptor agonist isoprenaline, despite being a partial agonist with respect to cAMP generation. GLUT4 translocation occurred independently of Akt and AMPK phosphorylation but was dependent on mTORC2. Furthermore, in contrast to isoprenaline, BRL37344 did not promote agonist-mediated desensitization and failed to recruit β-arrestin1/2 to the β2-adrenoceptor. In conclusion, BRL37344 improved glucose tolerance and increased glucose uptake into skeletal muscle in vivo and ex vivo through a β2-adrenoceptor-mediated mechanism independently of Akt. BRL37344 was a partial agonist with respect to cAMP, but a full agonist for glucose uptake, and importantly did not cause classical receptor desensitization or internalization of the receptor.

Background Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance. Results In this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions. Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK1/2 and p38 MAP kinases, and Ca2+ mobilization, supportive of low cell surface receptor expression. Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation. Conclusions This study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa. Electronic supplementary material The online version of this article (10.1186/s12885-019-5369-y) contains supplementary material, which is available to authorized users.

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 physiological role of vascular βStudies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. β-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of βAll three β-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The βAll three β-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but β

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.

Preterm infants frequently suffer cardiovascular compromise, with hypotension and/or low systemic blood flow, leading to tissue hypoxia-ischemia (HI). Many preterm infants respond inadequately to inotropic treatments using adrenergic agonists such as dobutamine (DB) or dopamine (DA). This may be because of altered cardiac adrenoceptor expression because of tissue HI or prolonged exposure to adrenergic agonists. We assessed the effects of severe HI with and without DB/DA treatment on cardiac adrenoceptor expression in preterm fetal sheep. Fetal sheep (93–95 days) exposed to sham surgery or severe HI induced by umbilical cord occlusion received intravenous DB or saline for 74 h (HI + DB, HI, Sham + DB, Sham). The HI groups were also compared with fetal sheep exposed to HI and DA. Fetal hearts were collected to determine β-adrenoceptor numbers using [125I]-cyanopindolol binding and mRNA expression of β1-, β2-, α1A-, α2A-, or α2B-adrenoceptors. The HI group had increased β-adrenoceptor numbers compared with all other groups in all four heart chambers ( P < 0.05). This increase in β-adrenoceptor numbers in the HI group was significantly reduced by DB infusion in all four heart chambers, but DA infusion in the HI group only reduced β-adrenoceptor numbers in the left atria and ventricle. DB alone did not affect β-adrenoceptor numbers in the sham animals. Changes in β1-adrenoceptor mRNA levels trended to parallel the binding results. We conclude that HI upregulates preterm fetal cardiac β-adrenoceptors, but prolonged exposure to adrenergic agonists downregulates adrenoceptors in the preterm heart exposed to HI and may underpin the frequent failure of inotropic therapy in preterm infants. NEW & NOTEWORTHY This is the first study, to our knowledge, on the effects of hypoxia-ischemia and adrenergic agonists on adrenoceptors in the preterm heart. In fetal sheep, we demonstrate that hypoxia-ischemia increases cardiac β-adrenoceptor numbers. However, exposure to both hypoxia-ischemia and adrenergic agonists (dobutamine or dopamine) reduces the increase in β-adrenoceptor numbers, which may underpin the inadequate response in human preterm infants to inotropic therapy using adrenergic agonists. Dobutamine alone does not affect the cardiac adrenoceptors in the sham animals.

Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4. Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.

The β3 -adrenoceptor was initially an attractive target for several pharmaceutical companies due to its high expression in rodent adipose tissue, where its activation resulted in decreased adiposity and improved metabolic outputs (such as glucose handling) in animal models of obesity and Type 2 diabetes. However, several drugs acting at the β3 -adrenoceptor failed in clinical trials. This was thought to be due to their lack of efficacy at the human receptor. Recently, mirabegron, a β3 -adrenoceptor agonist with human efficacy, was approved in North America, Europe, Japan and Australia for the treatment of overactive bladder syndrome. There are indications that mirabegron may act at other receptors/targets, but whether they have any clinical relevance is relatively unknown. Besides overactive bladder syndrome, mirabegron may have other uses such as in the treatment of heart failure or metabolic disease. This review gives an overview of the off-target effects of mirabegron and its potential use in the treatment of other diseases. Linked articles This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

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.

Background and Purpose Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. Experimental Approach Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. Key Results Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. Conclusion and Implications We have shown that while adrenergic agonists display bias at human α1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

Uptake of glucose into skeletal muscle and adipose tissue plays a vital role in metabolism and energy balance. Insulin released from β-islet cells of the pancreas promotes glucose uptake in these target tissues by stimulating translocation of GLUT4 transporters to the cell surface. This process is complex, involving signaling proteins including the mechanistic (or mammalian) target of rapamycin (mTOR) and Akt that intersect with multiple pathways controlling cell survival, growth and proliferation. mTOR exists in two forms, mTOR complex 1 (mTORC1), and mTOR complex 2 (mTORC2). mTORC1 has been intensively studied, acting as a key regulator of protein and lipid synthesis that integrates cellular nutrient availability and energy balance. Studies on mTORC2 have focused largely on its capacity to activate Akt by phosphorylation at Ser473, however recent findings demonstrate a novel role for mTORC2 in cellular glucose uptake. For example, agonists acting at β2-adrenoceptors (ARs) in skeletal muscle or β3-ARs in brown adipose tissue increase glucose uptake in vitro and in vivo via mechanisms dependent on mTORC2 but not Akt. In this review, we will focus on the signaling pathways downstream of β-ARs that promote glucose uptake in skeletal muscle and brown adipocytes, and will highlight how the insulin and adrenergic pathways converge and interact in these cells. The identification of insulin-independent mechanisms that promote glucose uptake should facilitate novel treatment strategies for metabolic disease.

Adrenoceptors play an important role in adipose tissue biology and physiology that includes regulating the synthesis and storage of triglycerides (lipogenesis), the breakdown of stored triglycerides (lipolysis), thermogenesis (heat production), glucose metabolism, and the secretion of adipocyte-derived hormones that can control whole-body energy homeostasis. These processes are regulated by the sympathetic nervous system through actions at different adrenoceptor subtypes expressed in adipose tissue depots. In this review, we have highlighted the role of adrenoceptor subtypes in white, brown, and brite adipocytes in both rodents and humans and have included detailed analysis of adrenoceptor expression in human adipose tissue and clonally derived adipocytes. We discuss important considerations when investigating adrenoceptor function in adipose tissue or adipocytes. 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.

Natural killer (NK) cells are innate lymphocytes that play a major role in immunosurveillance against tumor initiation and metastatic spread. The signals and checkpoints that regulate NK cell fitness and function in the tumor microenvironment are not well defined. Transforming growth factor-beta (TGF-beta) is a suppressor of NK cells that inhibits interleukin-15 (IL-15)-dependent signaling events and increases the abundance of receptors that promote tissue residency. Here, we showed that NK cells express the type I activin receptor ALK4, which, upon binding to its ligand activin-A, phosphorylated SMAD2/3 to suppress IL-15-mediated NK cell metabolism. Activin-A impaired human and mouse NK cell proliferation and reduced the production of granzyme B to impair tumor killing. Similar to TGF-beta, activin-A also induced SMAD2/3 phosphorylation and stimulated NK cells to increase their cell surface expression of several markers of ILC1 cells. Activin-A also induced these changes in TGF-beta receptor-deficient NK cells, suggesting that activin-A and TGF-beta stimulate independent pathways that drive SMAD2/3-mediated NK cell suppression. Last, inhibition of activin-A by follistatin substantially slowed orthotopic melanoma growth in mice. These data highlight the relevance of examining TGF-beta-independent SMAD2/3 signaling mechanisms as a therapeutic axis to relieve NK cell suppression and promote antitumor immunity.

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.

Endocannabinoids are products of dietary fatty acids that are modulated by an alteration in food intake levels. Overweight and obese individuals have substantially higher circulating levels of the arachidonic acid derived endocannabinoids, anandamide and 2-arachidonoyl glycerol, and show an altered pattern of cannabinoid receptor expression. These cannabinoid receptors are part of a large family of G protein coupled receptors (GPCRs). GPCRs are major therapeutic targets for various diseases within the cardiovascular, neurological, gastrointestinal, and endocrine systems, as well as metabolic disorders such as obesity and type 2 diabetes mellitus. Obesity is considered a state of chronic low-grade inflammation elicited by an immunological response. Interestingly, the newly deorphanized GPCR (GPR18), which is considered to be a putative cannabinoid receptor, is proposed to have an immunological function. In this review, the current scientific knowledge on GPR18 is explored including its localization, signaling pathways, and pharmacology. Importantly, the involvement of nutritional factors and potential dietary regulation of GPR18 and its (patho)physiological roles are described. Further research on this receptor and its regulation will enable a better understanding of the complex mechanisms of GPR18 and its potential as a novel therapeutic target for treating metabolic disorders.

There are two types of adipose tissue with distinct functions-white adipose tissue stores chemical energy as triglycerides, whereas brown adipose tissue consumes energy and releases heat (thermogenesis) in response to sympathetic nerve activity. In humans, treatments that promote greater brown adipose tissue deposition and/or activity would be highly beneficial in regimes aimed at reducing obesity. Adult humans have restricted populations of prototypical brown adipocytes in the neck and chest areas, but recent advances have established that adipocytes with similar properties, termed "brite" adipocytes, can be recruited in subcutaneous depots thought to be primarily white adipose tissue. These brite adipocytes express the protein machinery required for thermogenesis, but to assess brite adipocytes as viable therapeutic targets we need to understand how to promote conversion of white adipocytes to brite adipocytes and ways to increase optimal energy consumption and thermogenesis in these brite adipocytes. This can be accomplished by pharmacological and nutritional therapies to differing degrees, as reviewed in detail here.

Natural killer (NK) cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling and functional studies that the activating receptor DNAM-1 (CD226) identifies two distinct NK cell functional subsets: DNAM-1(+) and DNAM-1(-) NK cells. DNAM-1(+) NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1(-) NK cells that differentiate from DNAM-1(+) NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression.

Dysfunction of the endocannabinoid system (ECS) has been identified in metabolic disease. Cannabinoid receptor 1 (CB1) is abundantly expressed in the brain but also expressed in the periphery. Cannabinoid receptor 2 (CB2) is more abundant in the periphery, including the immune cells. In obesity, global antagonism of overexpressed CB1 reduces bodyweight but leads to centrally mediated adverse psychological outcomes. Emerging research in isolated cultured cells or tissues has demonstrated that targeting the endocannabinoid system in the periphery alleviates the pathologies associated with metabolic disease. Further, peripheral specific cannabinoid ligands can reverse aspects of the metabolic phenotype. This Keynote review will focus on current research on the functionality of peripheral modulation of the ECS for the treatment of obesity.

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

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

Background In this study, we tested five series of pyrazole-5-carboxamide compounds (n = 55) for activity against parasitic stages of the nematode Haemonchus contortus (barber’s pole worm), one of the most pathogenic parasites of ruminants. Methods In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility and development of H. contortus. Results Amongst the 55 compounds, we identified two compounds (designated a-15 and a-17) that reproducibly inhibit xL3 motility as well as L4 motility and development, with IC50 values ranging between ~3.4 and 55.6 μM. We studied the effect of these two ‘hit’ compounds on mitochondrial function by measuring oxygen consumption. This assessment showed that xL3s exposed to each of these compounds consumed significantly less oxygen and had less mitochondrial activity than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. Conclusions The present findings provide a sound basis for future work, aimed at identifying the targets of compounds a-15 and a-17 and establishing the modes of action of these chemicals in H. contortus. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2191-8) contains supplementary material, which is available to authorized users.

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express β3-adrenoceptors, and β3-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases β3-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β3-adrenoceptors following rosiglitazone treatment, which results in an increase in all β3-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α1-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.

The relaxin family peptide receptor 4 (RXFP4) is a G protein-coupled receptor (GPCR) expressed in the colorectum with emerging roles in metabolism and appetite regulation. It is activated by its cognate ligand insulin-like peptide 5 (INSL5) that is expressed in enteroendocrine L cells in the gut. Whether other evolutionarily related peptides such as relaxin-2, relaxin-3, or INSL3 activate RXFP4 signal transduction mechanisms with a pattern similar to or distinct from INSL5 is still unclear. In this study, we compare the signaling pathways activated by various relaxin family peptides to INSL5. We found that, like INSL5, relaxin-3 activated ERK1/2, p38MAPK, Akt, and S6RP phosphorylations leading to increased cell proliferation and also caused GRK and β-arrestin-mediated receptor internalization. Interestingly, relaxin-3 was slightly more potent than INSL5 in ERK1/2 and Akt phosphorylations, but both peptides were almost equipotent in adenylyl cyclase inhibition, S6RP phosphorylation, and cell proliferation. In addition, relaxin-3 showed greater efficacy only in Akt phosphorylation but not in the other pathways investigated. In contrast, no signaling activity or receptor internalization mechanisms were observed following relaxin-2 and INSL3. In conclusion, relaxin-3 is a high-efficacy agonist at RXFP4 with a comparable signal transduction profile to INSL5.

Background and purpose Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide-bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide RXFP4 receptor that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4 receptors. Experimental approach INSL5/RXFP4 receptor signalling was investigated using AlphaScreen® proximity assays. Recruitment of Gαi/o proteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gαi/o C351I mutants. Cell proliferation was studied with bromodeoxyuridine. RXFP4 receptor interactions with β-arrestins, GPCR kinase 2 (GRK2), KRas and Rab5a was assessed with real-time BRET. Gene expression was investigated using real-time quantitative PCR. Insulin release was measured using HTRF and intracellular Ca2+ flux monitored in a Flexstation® using Fluo-4-AM. Key results INSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt Ser473 , Akt Thr308 and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGαoA , mGαoB and mGαi2 and to a lesser extent mGαi1 and mGαi3 . RXFP4 receptors interacted with GRK2 and β-arrestins, moved towards Rab5a and away from KRas, indicating internalisation following receptor activation. INSL5 inhibited glucose-stimulated insulin secretion and Ca2+ mobilisation in MIN6 insulinoma cells and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells. Conclusions and implications Knowledge of signalling pathways activated by INSL5 at RXFP4 receptors is essential for understanding the biological roles of this novel gut hormone. Linked articles This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

International audience; The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

BACKGROUND AND PURPOSE β-Adrenoceptor stimulation induces glucose uptake in several insulin-sensitive tissues by poorly understood mechanisms. EXPERIMENTAL APPROACH We used a model system in CHO-K1 cells expressing the human β2-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved. KEY RESULTS In CHO-K1 cells, there was no response to β-adrenoceptor agonists. The introduction of β2-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to β-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and β2-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to β2-adrenoceptor stimulation. Truncation of the C-terminus of the β2-adrenoceptor at position 349 to remove known phosphorylation sites for GPCR kinases (GRKs) or at position 344 to remove an additional PKA site together with the GRK phosphorylation sites did not significantly affect cAMP accumulation but decreased β2-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the βARKct construct inhibited β2-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing β2-adrenoceptors lacking phosphorylation sites for GRK or PKA demonstrated that the GRK sites, but not the PKA sites, were necessary for GLUT4 translocation. CONCLUSIONS AND IMPLICATIONS Glucose uptake in response to activation of β2-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the β2-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.

With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via β3-adrenergic receptors. However, vast majorities of β3-adrenergic agonists have so far not been able to stimulate human β3-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by β1-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking β3-adrenergic receptors. Wild-type and β3-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. β3-knockout mice were able to survive both acute and prolonged cold exposure due to activation of β1-adrenergic receptors. Thus, in the absence of β3-adrenergic receptors, β1-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either β1-adrenergic receptors or β3-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, β1-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.

Background and purpose β(1) -, β(2) - and β(3) -adrenoceptors determined by functional, binding and reverse transcription polymerase chain reaction (RT-PCR) studies are present in chick astrocytes and activation of β(2) - or β(3) -adrenoceptors increase glucose uptake. The aims of the present study are to identify which β-adrenoceptor subtypes are present in mouse astrocytes, the signal transduction mechanisms involved and whether β-adrenoceptor stimulation regulates glucose uptake. Experimental approach Astrocytes were prepared from four mouse strains: FVB/N, DBA/1 crossed with C57BL/6J, β(3) -adrenoceptor knockout and β(1) β(2) -adrenoceptor knockout mice. RT-PCR and radioligand binding studies were used to determine β-adrenoceptor expression. Glucose uptake and cAMP were assayed to elucidate the signalling pathways involved. Key results mRNAs for all three β-adrenoceptors were identified in astrocytes from wild-type mice. Radioligand binding studies identified that β(1) - and β(3) -adrenoceptors were predominant. cAMP studies showed that β(1) - and β(2) -adrenoceptors coupled to G(s) whereas β(3) -adrenoceptors coupled to both G(s) and G(i) . However, activation of any of the three β-adrenoceptors increased glucose uptake in mouse astrocytes. Interestingly, there was no functional compensation for receptor subtype loss in knockout animals. Conclusions and implications This study demonstrates that although β(1) -adrenoceptors are the predominant β-adrenoceptor in mouse astrocytes and are primarily responsible for cAMP production in response to β-adrenoceptor stimulation, β(3) -adrenoceptors are also present in mouse astrocytes and activation of β(2) - and β(3) -adrenoceptors increases glucose uptake in mouse astrocytes.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.