Molecular mechanism of GPCRs mediating metabolic responses – The role of adrenoceptors in glucose metabolism

Mukaida, Saori (2019) Molecular mechanism of GPCRs mediating metabolic responses – The role of adrenoceptors in glucose metabolism. PhD thesis, University of Nottingham, Monash University.

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Abstract

Insulin-stimulated glucose uptake requires an intricate network of proteins including phosphoinositide 3-kinase, Akt and glucose transporter 4 (GLUT4), and is key to maintaining whole body glucose homeostasis. Dysfunction of insulin action causes diminished glucose uptake/utilization in insulin target tissues. Adrenoceptors (ARs), members of the G protein-coupled receptor superfamily, are also involved in glucose metabolism, and activation of α1A-ARs, β2-ARs and β3-ARs increases glucose uptake in cardiomyocytes, skeletal muscle and brown adipocytes, respectively. However, there is a knowledge gap on the mechanisms involved in AR-mediated glucose uptake, and how they differ from that of insulin, which may be important in disease context (such as type 2 diabetes) where the insulin-signaling pathway is down-regulated.

We examined the differences in signalling profiles between two β2-AR agonists, isoprenaline and BRL37344, in skeletal muscle. BRL37344 increased skeletal muscle glucose uptake in vivo and improved glucose tolerance in vivo in insulin-resistant mice, showing potential anti-diabetic effects. In skeletal muscle cells in vitro, BRL37344 increased GLUT4 translocation and glucose uptake with a similar potency and efficacy to that of isoprenaline. However, BRL37344 was a weak partial agonist for cAMP production and failed to desensitise the β2-AR, as it was unable to promote β-arrestin1/2 binding to the β2-AR. The role of GRK (which has a primary role in receptor desensitisation) was further investigated in a recombinant system using several mutant β2-ARs partially or completely lacking putative GRK/PKA phosphorylation sites, since siRNA directed against GRK2 abolished β2-AR mediated glucose uptake. Attenuation of GRK phosphorylation sites disrupted isoprenaline-stimulated receptor internalisation but did not affect GLUT4 translocation/glucose uptake. In contrast, BRL37344 stimulation did not induce internalisation and did not alter GLUT4 translocation/glucose uptake in the presence or absence of GRK phosphorylation sites. This suggested that GRK phosphorylation sites in β2-AR C-terminal do not affect β2-AR mediated glucose uptake, and that GRK may function in a novel manner to regulate this pathway.

Stimulation of cardiomyocyte α1A-ARs prevents pathologic remodeling in heart failure, potentially due to increased glucose uptake and protein synthesis. In a recombinant system, we assessed the signalling pathways activated by two α1A-AR agonists: A61603 and dabuzalgron. A61603 increased cAMP production, which is partially involved in glucose uptake. In contrast, dabuzalgron increased glucose uptake independently of cAMP. Both A61603 and dabuzalgron were full agonists for glucose uptake and extracellular signal-regulated kinases1/2 phosphorylation (associated with cell survival), but dabuzalgron was a partial agonist for Ca2+ mobilization (contractility), S6 ribosomal protein phosphorylation (hypertrophy), and N-Myc Downstream Regulated 1 phosphorylation (downstream of mTORC2). This suggests that dabuzalgron may be a biased agonist with respect to cell survival and glucose uptake in contrast to contractility and hypertrophy. These findings establish a novel link between α1A-ARs, cAMP and glucose uptake, and provide an improved framework for examining the utility of α1A-AR agonists as cardioprotective agents. These studies collectively advance our basic understanding of how ARs can increase glucose uptake.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Holliday, Nicholas
Hutchinson, Dana
Sato, Masaaki
Summers, Roger
Evans, Bronwyn
Keywords: Glucose uptake, Adrenoceptor
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56997
Depositing User: Mukaida, Saori
Date Deposited: 21 Oct 2019 10:46
Last Modified: 06 May 2020 13:46
URI: http://eprints.nottingham.ac.uk/id/eprint/56997

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