Publications by authors named "Ling Yeong Chia"

9 Publications

  • Page 1 of 1

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

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

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

Isolation, Purification, Generation, and Culture of Osteocytes.

Methods Mol Biol 2019 ;1914:39-51

Department of Medicine, St. Vincent's Hospital Melbourne, The University of Melbourne, Melbourne, VIC, Australia.

Osteocytes reside within bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been difficult to study in vitro because they are difficult to extract and purify, and do not retain their phenotype in standard culture conditions. However, new techniques for this purpose are emerging. This chapter will describe three methods we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic-lineage depletion, (2) differentiating cultured osteoblasts (or osteoblast cell lines) until they reach a stage of osteocytic gene expression, and (3) using the Ocy454 osteocyte-like cell line.
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http://dx.doi.org/10.1007/978-1-4939-8997-3_3DOI Listing
June 2019

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

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

Androgen Action via the Androgen Receptor in Neurons Within the Brain Positively Regulates Muscle Mass in Male Mice.

Endocrinology 2017 10;158(10):3684-3695

Department of Medicine, Austin Health, University of Melbourne, Heidelberg, Victoria 3084, Australia.

Although it is well established that exogenous androgens have anabolic effects on skeletal muscle mass in humans and mice, data from muscle-specific androgen receptor (AR) knockout (ARKO) mice indicate that myocytic expression of the AR is dispensable for hind-limb muscle mass accrual in males. To identify possible indirect actions of androgens via the AR in neurons to regulate muscle, we generated neuron-ARKO mice in which the dominant DNA binding-dependent actions of the AR are deleted in neurons of the cortex, forebrain, hypothalamus, and olfactory bulb. Serum testosterone and luteinizing hormone levels were elevated twofold in neuron-ARKO males compared with wild-type littermates due to disruption of negative feedback to the hypothalamic-pituitary-gonadal axis. Despite this increase in serum testosterone levels, which was expected to increase muscle mass, the mass of the mixed-fiber gastrocnemius (Gast) and the fast-twitch fiber extensor digitorum longus hind-limb muscles was decreased by 10% in neuron-ARKOs at 12 weeks of age, whereas muscle strength and fatigue of the Gast were unaffected. The mass of the soleus muscle, however, which consists of a high proportion of slow-twitch fibers, was unaffected in neuron-ARKOs, demonstrating a stimulatory action of androgens via the AR in neurons to increase the mass of fast-twitch hind-limb muscles. Furthermore, neuron-ARKOs displayed reductions in voluntary and involuntary physical activity by up to 60%. These data provide evidence for a role of androgens via the AR in neurons to positively regulate fast-twitch hind-limb muscle mass and physical activity in male mice.
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http://dx.doi.org/10.1210/en.2017-00470DOI Listing
October 2017

Isolation and gene expression of haematopoietic-cell-free preparations of highly purified murine osteocytes.

Bone 2015 Mar 15;72:34-42. Epub 2014 Nov 15.

St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia; Department of Medicine at St. Vincent's Hospital, The University of Melbourne, Fitzroy, Victoria, Australia. Electronic address:

To define their gene expression and function, osteocytes are commonly isolated and purified by fluorescence-activated cell sorting (FACS) from mice expressing GFP directed by the dentin matrix protein 1 (Dmp1) promoter (DMP1-GFP). These cells express mRNA for osteocyte genes, including sclerostin (Sost) and Dmp1, and genes associated with the osteoclast phenotype: Dcstamp, Oscar, Cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAP/Acp5) and calcitonin receptor (Calcr). This suggests either that osteoclasts and osteocytes share genes and functions or that DMP1-GFP(+) preparations contain haematopoietic osteoclasts. To resolve this we stained DMP1-GFP cells for haematopoietic lineage (Lin) surface markers (CD2, CD3e, CD4, CD45, CD5, CD8, CD11b, B220, Gr1, Ter119) and CD31. Lin(-)CD31(-) (Lin(-)) and Lin(+)CD31(+) (Lin(+)) populations were analysed for GFP, and the four resulting populations assessed by quantitative real-time PCR. Lin(-)GFP(+) cells expressed mRNAs for Sost, Dmp1, and Mepe, confirming their osteocyte identity. Dcstamp and Oscar mRNAs were restricted to haematopoietic (Lin(+)) cells, but Calcr, Ctsk and Acp5 were readily detected in purified osteocytes (Lin(-)GFP(+)). The capacity of these purified osteocytes to support osteoclastogenesis was assessed: no TRAP+ cells with >2 nuclei were formed when purified osteocytes were cultured with bone marrow macrophages and stimulated with 1,25-dihydroxyvitamin-D3/prostaglandin E2. Lin(-)GFP(+) osteocytes also expressed lower levels of Tnfsf11 (RANKL) mRNA than the osteoblast-enriched population (Lin(-)GFP(-)). This demonstrates the importance of haematopoietic depletion in generating highly purified osteocytes and shows that osteocytes express Acp5, Ctsk and Calcr, but not other osteoclast markers, and do not fully support osteoclast formation in vitro.
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http://dx.doi.org/10.1016/j.bone.2014.11.005DOI Listing
March 2015

Decline in calcitonin receptor expression in osteocytes with age.

J Endocrinol 2014 May 22;221(2):181-91. Epub 2014 Apr 22.

St Vincent's Institute, and Department of Medicine, at St Vincent's Hospital, The University of Melbourne, 9 Princes Street, Fitzroy, Victoria 3065, Australia Nordic Bioscience Inc., Copenhagen, Denmark.

We have previously shown that co-administration of the transient osteoclast inhibitor, salmon calcitonin (sCT), blunts the anabolic effect of parathyroid hormone (PTH) in young rats and increases osteocytic expression of the bone formation inhibitor sclerostin (Sost). To determine whether this also occurs in adult animals, we co-administered sCT with PTH to 6-month-old sham-operated (SHAM) and ovariectomised (OVX) rats. While sCT reduced the stimulatory effect of PTH on serum amino-terminal propeptide of type 1 procollagen levels, in contrast to its influence in young rats, sCT did not reduce the anabolic effect of PTH on femoral bone mineral density, tibial trabecular bone volume or bone formation rate in 6-month-old SHAM or OVX rats. Quantitative real-time PCR analysis of femoral metaphyses collected 1 and 4 h after a single PTH injection confirmed a significant increase in mRNA levels for interleukin 6 (Il6) and ephrinB2 (EfnB2), and a significant reduction in Sost and dentin matrix protein-1 (Dmp1) in response to PTH. However, in contrast to observations in young rats, these effects were not modified by co-administration of sCT, nor did sCT significantly modify Sost, Dmp1, or matrix extracellular phosphoglycoprotein (Mepe) mRNA levels. Furthermore, while CT receptor (CTR) mRNA (Calcr) was readily detected in GFP+ osteocytes isolated from young (3-week-old) DMP1-GFP mice, Calcr levels in osteocytes declined as mice aged, reaching levels that were undetectable in long bone at 49 weeks of age. These data indicate that osteocyte-mediated responses to CT are most likely to be of physiological relevance in young rodents.
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http://dx.doi.org/10.1530/JOE-13-0524DOI Listing
May 2014