Publications by authors named "Dana S Hutchinson"

58 Publications

GPR55 regulates the responsiveness to, but does not dimerise with, α-adrenoceptors.

Biochem Pharmacol 2021 May 1;188:114560. Epub 2021 May 1.

Cardiometabolic Health Research, School of Pharmacy and Life Sciences, Robert Gordon University, Sir Ian Wood Building, Aberdeen AB10 7GJ, UK.

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-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α-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 α-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α-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 (α-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 α-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α-AR failed to dimerize, pharmacological blockade of GPR55 altered α-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α-AR in the cardiovascular system.
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http://dx.doi.org/10.1016/j.bcp.2021.114560DOI Listing
May 2021

Effect of β /β -adrenoceptor blockade on β -adrenoceptor activity in the rat cremaster muscle artery.

Br J Pharmacol 2021 Apr 27;178(8):1789-1804. Epub 2021 Feb 27.

Physiology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

Background And Purpose: The physiological role of vascular β -adrenoceptors is not fully understood. Recent evidence suggests cardiac β -adrenoceptors are functionally effective after down-regulation of β /β -adrenoceptors. The functional interaction between the β -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 β -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 β -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 β -adrenoceptor agonist SR 58611A and the nonselective β-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of β -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 β -adrenoceptor antagonist L 748,337 (1 μM) inhibited vasodilation caused by β -adrenoceptor agonists (in the presence of β -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 β -adrenoceptor mediated vasodilation was only evident after blockade of β -adrenoceptors. This suggests constitutive β -adrenoceptor activity inhibits β -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.
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http://dx.doi.org/10.1111/bph.15398DOI Listing
April 2021

The metabolic effects of mirabegron are mediated primarily by β -adrenoceptors.

Pharmacol Res Perspect 2020 10;8(5):e00643

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

The β -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, [ H]-2-deoxyglucose uptake, cellular glycolysis, and O 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 β -adrenoceptors as they were largely absent in adipocytes derived from β -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 β -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 β -adrenoceptor.
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http://dx.doi.org/10.1002/prp2.643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437350PMC
October 2020

Acute β-adrenoceptor mediated glucose clearance in brown adipose tissue; a distinct pathway independent of functional insulin signaling.

Mol Metab 2019 12 18;30:240-249. Epub 2019 Oct 18.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91, Stockholm, Sweden. Electronic address:

Objective: β-adrenoceptor mediated activation of brown adipose tissue (BAT) has been associated with improvements in metabolic health in models of type 2 diabetes and obesity due to its unique ability to increase whole body energy expenditure, and rate of glucose and free fatty acid disposal. While the thermogenic arm of this phenomenon has been studied in great detail, the underlying mechanisms involved in β-adrenoceptor mediated glucose uptake in BAT are relatively understudied. As β-adrenoceptor agonist administration results in increased hepatic gluconeogenesis that can consequently result in secondary pancreatic insulin release, there is uncertainty regarding the importance of insulin and the subsequent activation of its downstream effectors in mediating β-adrenoceptor stimulated glucose uptake in BAT. Therefore, in this study, we made an effort to discriminate between the two pathways and address whether the insulin signaling pathway is dispensable for the effects of β-adrenoceptor activation on glucose uptake in BAT.

Methods: Using a specific inhibitor of phosphoinositide 3-kinase α (PI3Kα), which effectively inhibits the insulin signaling pathway, we examined the effects of various β-adrenoceptor agonists, including the physiological endogenous agonist norepinephrine on glucose uptake and respiration in mouse brown adipocytes in vitro and on glucose clearance in mice in vivo.

Results: PI3Kα inhibition in mouse primary brown adipocytes in vitro, did not inhibit β-adrenoceptor stimulated glucose uptake, GLUT1 synthesis, GLUT1 translocation or respiration. Furthermore, β-adrenoceptor mediated glucose clearance in vivo did not require insulin or Akt activation but was attenuated upon administration of a β-adrenoceptor antagonist.

Conclusions: We conclude that the β-adrenergic pathway is still functionally intact upon the inhibition of PI3Kα, showing that the activation of downstream insulin effectors is not required for the acute effects of β-adrenoceptor agonists on glucose homeostasis or thermogenesis.
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http://dx.doi.org/10.1016/j.molmet.2019.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838983PMC
December 2019

Therapeutic blockade of activin-A improves NK cell function and antitumor immunity.

Sci Signal 2019 08 27;12(596). Epub 2019 Aug 27.

Division of Molecular Immunology, The Walter and Eliza Hall Institute of Medical Research and Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.

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-β (TGF-β) 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-β, 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-β receptor-deficient NK cells, suggesting that activin-A and TGF-β 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-β-independent SMAD2/3 signaling mechanisms as a therapeutic axis to relieve NK cell suppression and promote antitumor immunity.
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http://dx.doi.org/10.1126/scisignal.aat7527DOI Listing
August 2019

Adrenoceptors in white, brown, and brite adipocytes.

Br J Pharmacol 2019 07 7;176(14):2416-2432. Epub 2019 Apr 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1111/bph.14631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592855PMC
July 2019

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas.

BMC Cancer 2019 Feb 18;19(1):157. Epub 2019 Feb 18.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia.

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 ERK and p38 MAP kinases, and Ca 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.
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http://dx.doi.org/10.1186/s12885-019-5369-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379965PMC
February 2019

Adrenoceptor regulation of the mechanistic target of rapamycin in muscle and adipose tissue.

Br J Pharmacol 2019 07 7;176(14):2433-2448. Epub 2019 Apr 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.

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.
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http://dx.doi.org/10.1111/bph.14616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592864PMC
July 2019

Effects of hypoxia-ischemia and inotropes on expression of cardiac adrenoceptors in the preterm fetal sheep.

J Appl Physiol (1985) 2018 11 23;125(5):1368-1377. Epub 2018 Aug 23.

The Ritchie Centre, The Hudson Institute of Medical Research , Melbourne , Australia.

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 [I]-cyanopindolol binding and mRNA expression of β-, β-, α-, α-, or α-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 β-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.
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http://dx.doi.org/10.1152/japplphysiol.00472.2018DOI Listing
November 2018

Rosiglitazone and a β-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture.

Front Endocrinol (Lausanne) 2018 30;9:249. Epub 2018 May 30.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

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 β-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 β-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β-adrenoceptor expression and provides an improved window for detection of β-adrenoceptor responses.
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http://dx.doi.org/10.3389/fendo.2018.00249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992408PMC
May 2018

INSL5 activates multiple signalling pathways and regulates GLP-1 secretion in NCI-H716 cells.

J Mol Endocrinol 2018 04;60(3):213-224

Drug Discovery BiologyMonash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

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 hybridisation that 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 Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr/Tyr), AKT (Thr and Ser) and S6RP (Ser) and inhibited cAMP production but did not stimulate Ca 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 Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.
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http://dx.doi.org/10.1530/JME-17-0152DOI Listing
April 2018

Peripheral modulation of the endocannabinoid system in metabolic disease.

Drug Discov Today 2018 03 10;23(3):592-604. Epub 2018 Jan 10.

Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, Melbourne, VIC, Australia; School of Science, Menzies Health Institute Queensland, Griffith University, Nathan, QLD, Australia. Electronic address:

Dysfunction of the endocannabinoid system (ECS) has been identified in metabolic disease. Cannabinoid receptor 1 (CB) is abundantly expressed in the brain but also expressed in the periphery. Cannabinoid receptor 2 (CB) is more abundant in the periphery, including the immune cells. In obesity, global antagonism of overexpressed CB 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.
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http://dx.doi.org/10.1016/j.drudis.2018.01.029DOI Listing
March 2018

α-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

Biochem Pharmacol 2018 02 24;148:27-40. Epub 2017 Nov 24.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address:

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-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α-AR signalling in CHO-K1 cells co-expressing the human α-AR and GLUT4 (CHOαGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOαGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α-AR agonists. Our findings identify a novel link between the α-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α-AR selective agonists as tools in the treatment of cardiac dysfunction.
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http://dx.doi.org/10.1016/j.bcp.2017.11.016DOI Listing
February 2018

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes, increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake.

Cell Signal 2018 Jan 29;42:54-66. Epub 2017 Sep 29.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Monash University, Parkville, Victoria 3052, Australia; Department of Pharmacology, 9 Ancora Imparo Way, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

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 β-adrenoceptors, and β-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 β-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β-adrenoceptors following rosiglitazone treatment, which results in an increase in all β-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α-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.
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http://dx.doi.org/10.1016/j.cellsig.2017.09.023DOI Listing
January 2018

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.

Sci Rep 2017 06 7;7(1):2968. Epub 2017 Jun 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

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 Gα and Gα but weak coupling to Gα. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.
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http://dx.doi.org/10.1038/s41598-017-02916-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462828PMC
June 2017

Factors influencing biased agonism in recombinant cells expressing the human α -adrenoceptor.

Br J Pharmacol 2017 Jul 10;174(14):2318-2333. Epub 2017 Jun 10.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

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 α -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 Ca 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 Ca mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca 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-HT receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca 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 α -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.
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http://dx.doi.org/10.1111/bph.13837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481649PMC
July 2017

Adrenoceptors promote glucose uptake into adipocytes and muscle by an insulin-independent signaling pathway involving mechanistic target of rapamycin complex 2.

Pharmacol Res 2017 02 23;116:87-92. Epub 2016 Dec 23.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

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 β-adrenoceptors (ARs) in skeletal muscle or β-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.
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http://dx.doi.org/10.1016/j.phrs.2016.12.022DOI Listing
February 2017

The actions of relaxin family peptides on signal transduction pathways activated by the relaxin family peptide receptor RXFP4.

Naunyn Schmiedebergs Arch Pharmacol 2017 Jan 26;390(1):105-111. Epub 2016 Nov 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

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.
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http://dx.doi.org/10.1007/s00210-016-1321-8DOI Listing
January 2017

Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

Br J Pharmacol 2017 05 13;174(10):1077-1089. Epub 2016 Jul 13.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

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 Gα proteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gα 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 Ca 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 Ser , Akt Thr and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGα , mGα and mGα and to a lesser extent mGα and mGα . 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 Ca 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.
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http://dx.doi.org/10.1111/bph.13522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406383PMC
May 2017

CIS is a potent checkpoint in NK cell-mediated tumor immunity.

Nat Immunol 2016 07 23;17(7):816-24. Epub 2016 May 23.

Immunology in Cancer and Infection Laboratory QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

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.
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http://dx.doi.org/10.1038/ni.3470DOI Listing
July 2016

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15.

Immunity 2016 Jan 12;44(1):103-115. Epub 2016 Jan 12.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.immuni.2015.12.007DOI Listing
January 2016

G protein coupled receptor 18: A potential role for endocannabinoid signaling in metabolic dysfunction.

Mol Nutr Food Res 2016 Jan 22;60(1):92-102. Epub 2015 Sep 22.

Centre for Chronic Disease Prevention and Management, College of Health & Biomedicine, Victoria University, Melbourne, VIC, Australia.

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.
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http://dx.doi.org/10.1002/mnfr.201500449DOI Listing
January 2016

Could burning fat start with a brite spark? Pharmacological and nutritional ways to promote thermogenesis.

Mol Nutr Food Res 2016 Jan 27;60(1):18-42. Epub 2015 Aug 27.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

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.
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http://dx.doi.org/10.1002/mnfr.201500251DOI Listing
January 2016

DNAM-1 expression marks an alternative program of NK cell maturation.

Cell Rep 2015 Apr 26;11(1):85-97. Epub 2015 Mar 26.

Immunology in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia; School of Medicine, University of Queensland, Herston, QLD 4006, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2015.03.006DOI Listing
April 2015

Glucose uptake in brown fat cells is dependent on mTOR complex 2-promoted GLUT1 translocation.

J Cell Biol 2014 Nov;207(3):365-74

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE -0691 Stockholm, Sweden

Brown adipose tissue is the primary site for thermogenesis and can consume, in addition to free fatty acids, a very high amount of glucose from the blood, which can both acutely and chronically affect glucose homeostasis. Here, we show that mechanistic target of rapamycin (mTOR) complex 2 has a novel role in β3-adrenoceptor-stimulated glucose uptake in brown adipose tissue. We show that β3-adrenoceptors stimulate glucose uptake in brown adipose tissue via a signaling pathway that is comprised of two different parts: one part dependent on cAMP-mediated increases in GLUT1 transcription and de novo synthesis of GLUT1 and another part dependent on mTOR complex 2-stimulated translocation of newly synthesized GLUT1 to the plasma membrane, leading to increased glucose uptake. Both parts are essential for β3-adrenoceptor-stimulated glucose uptake. Importantly, the effect of β3-adrenoceptor on mTOR complex 2 is independent of the classical insulin-phosphoinositide 3-kinase-Akt pathway, highlighting a novel mechanism of mTOR complex 2 activation.
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http://dx.doi.org/10.1083/jcb.201403080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226734PMC
November 2014

Improving type 2 diabetes through a distinct adrenergic signaling pathway involving mTORC2 that mediates glucose uptake in skeletal muscle.

Diabetes 2014 Dec 9;63(12):4115-29. Epub 2014 Jul 9.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

There is an increasing worldwide epidemic of type 2 diabetes that poses major health problems. We have identified a novel physiological system that increases glucose uptake in skeletal muscle but not in white adipocytes. Activation of this system improves glucose tolerance in Goto-Kakizaki rats or mice fed a high-fat diet, which are established models for type 2 diabetes. The pathway involves activation of β2-adrenoceptors that increase cAMP levels and activate cAMP-dependent protein kinase, which phosphorylates mammalian target of rapamycin complex 2 (mTORC2) at S2481. The active mTORC2 causes translocation of GLUT4 to the plasma membrane and glucose uptake without the involvement of Akt or AS160. Stimulation of glucose uptake into skeletal muscle after activation of the sympathetic nervous system is likely to be of high physiological relevance because mTORC2 activation was observed at the cellular, tissue, and whole-animal level in rodent and human systems. This signaling pathway provides new opportunities for the treatment of type 2 diabetes.
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http://dx.doi.org/10.2337/db13-1860DOI Listing
December 2014

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

PLoS One 2013 25;8(7):e68841. Epub 2013 Jul 25.

Commissariat à l'Énergie Atomique Et Aux Énergies Alternatives, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif sur Yvette, France.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723878PMC
March 2014

β2-Adrenoceptor-mediated regulation of glucose uptake in skeletal muscle--ligand-directed signalling or a reflection of system complexity?

Naunyn Schmiedebergs Arch Pharmacol 2013 Sep 9;386(9):757-60. Epub 2013 May 9.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 399 Royal Parade, Parkville, Melbourne, VIC, 3052, Australia.

The capacity of G protein-coupled receptors (GPCRs) to activate multiple G protein isoforms and additional effectors such as β-arrestins has become a well-established paradigm and provides the basis for developing drugs that preferentially activate beneficial signalling pathways. There are many published examples of ligand-directed signalling, and recent studies have provided direct evidence that different agonists stabilise distinct GPCR conformations. This field is rapidly evolving, but a key question is whether signalling bias observed in heterologous cell expression systems can be translated to physiological systems of therapeutic relevance. The paper by Ngala et al. in this issue of the journal addresses the capacity of agonists acting at the β2-adrenoceptor to engender signalling bias in relation to glucose uptake in isolated skeletal muscle, an area of considerable potential interest in targeting insulin-independent pathways for the treatment of type 2 diabetes. The authors show that clenbuterol and BRL37344 have opposite effects on glucose uptake, despite both having agonist actions at β2-adrenoceptors. This study underlines some of the obstacles associated with studies in a complex physiological system but nonetheless highlights the need to consider signalling bias in the relevant target tissue when developing novel drugs.
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http://dx.doi.org/10.1007/s00210-013-0879-7DOI Listing
September 2013

Rapid turnover of glycogen in memory formation.

Neurochem Res 2012 Nov 5;37(11):2456-63. Epub 2012 Jun 5.

Monash Institute of Pharmaceutical Sciences, Parkville, VIC 3052, Australia.

The influence of noradrenaline acting at α(2)-AR and β(2)-ARs on the turnover of glycogen after learning has been investigated. The role of glycogen turnover in memory formation was examined using weakly-reinforced, single trial bead discrimination training in day-old domestic chickens. This study follows our previous work that focused on the need for glycogen breakdown (glycogenolysis) during learning. Inhibition of glycogenolysis by 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prevented the consolidation of strongly-reinforced learning and inhibited memory. The action of DAB could be prevented by stimulating glycogenolysis with the selective β(2)-AR agonist, zinterol. Stimulation of α(2)-ARs has been shown to lead to an increase in the turnover and synthesis of glycogen. In the present study, we examined the effect of inhibition of α(2)-AR stimulated glycogen turnover (measured as(14)C-glucose incorporation into glycogen) on the ability of zinterol to promote the consolidation of weakly reinforced memory. In astrocytes, the selective α(2)-AR agonist clonidine stimulated (14)C-glucose incorporation into glycogen in chick astrocytes and this was inhibited by the selective α(2)-AR antagonist, ARC239. The critical importance of the timing of ARC239 injection relative to training and intracerebral administration of zinterol was examined. It is concluded that our data provides evidence for a readily accessible labile pool of glycogen in brain astrocytes. If glycogen synthesis is inhibited, the can be depleted within 10 min, thus preventing zinterol from promoting consolidation.
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http://dx.doi.org/10.1007/s11064-012-0805-2DOI Listing
November 2012