BRL37344 stimulates glucose uptake into skeletal muscle by β2-adrenoceptor mediated translocation of GLUT4 without causing receptor desensitization

[Speaker] Saori Mukaida:1
[Co-author] Masaaki Sato:1, Michelle L Halls:1, Martina Kocan:1, Bronwyn A Evans:1, Roger J Summers:1, Tore Bengtsson:2, Dana S Hutchinson:1
1:Monash Institute of Pharmaceutical sciences, Monash university, Australia, 2:The Wenner-Gren Institute (MBW), Stockholm University, Sweden

Background: Cyclic AMP (cAMP) is a key second messenger involved in β2-adrenoceptor (β2-AR) mediated glucose uptake in skeletal muscle, but in vivo causes adverse effects including altered vasoreactivity and hypertrophy. It has been suggested that BRL37344 (BRL), a dual β23-AR agonist originally developed for the treatment of obesity, increases skeletal muscle glucose uptake via β2-ARs independently of cAMP, but the mechanism has not been rigorously examined (Nevzorova et al 2002). This study examines the signalling mechanisms activated by isoprenaline (Iso) and BRL to increase glucose uptake in L6 skeletal muscle cells.
Methods: We measured glucose uptake with 3H 2-deoxy-glucose; cAMP production was measured using a LANCE cAMP assay and Förster resonance energy transfer (FRET) to measure cAMP at the plasma membrane (pmEpac2) or in the cytoplasm (cytoEpac2); Bioluminescence resonance energy transfer (BRET) was performed to measure receptor interaction with β-arrestin, or receptor internalization as measured with dissociation with Kras (a membrane localised protein). Translocation of exofacially myc-tagged GLUT4 was determined by immunocytochemistry using non-permeabilized L6-GLUT4myc cells.
Results: In L6 cells, BRL increased glucose uptake with a similar potency and efficacy to that of Iso (pEC50 7.41±0.2 and 7.45±0.3; Emax 168.1±4.6 and 186.8±7.9 respectively, n=6), with immunohistochemical studies showing that both BRL and Iso increased GLUT4 translocation to the plasma membrane. BRL was however a partial agonist when measuring global cAMP levels (Iso pEC50 8.44±;0.1 Emax 15.5±0.5pmol cAMP/well; BRL pEC50 6.57±0.1 Emax 6.9±0.3pmol cAMP/well; n=4-6). FRET studies showed that BRL caused significantly less activation of the plasma membrane cAMP sensor pmEpac2 and the cytoplasmic cAMP sensor cytoEpac2 as compared to Iso. BRET studies showed that Iso, but not BRL, caused interaction between the β2-AR and β-arrestin1/2, movement of the receptor away from Kras, and receptor desensitisation.
Conclusions: Our results show that BRL is a partial β2-AR agonist, but a full agonist when measuring increases in glucose uptake. BRL does not result in receptor desensitization or internalization of the receptor.

Nevzorova, J., et al. (2002) Br J Pharmacol, 137:9-18.

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