Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and beta2-adrenoceptor mechanisms.

British Journal of Pharmacology (2008) 155, 395–406 & 2008 Macmillan Publishers Limited All rights reserved 0007– 1188/08 $32.00 www.brjpharmacol.org RESEARCH PAPER Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and b2-adrenoceptor mechanisms RA Ngala1, J O’Dowd, SJ Wang2, A Agarwal3, C Stocker, MA Cawthorne and JRS Arch Clore Laboratory, University of Buckingham, Buckingham, UK Background and purpose: Picomolar concentrations of the b3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via b2-adrenoceptors. Effects of BRL37344 and b2-adrenoceptor agonists are compared. Experimental approach: Mouse soleus muscles were incubated with 2-deoxy[1-14C]-glucose, [1-14C]-palmitate or [2-14C]pyruvate, and BRL37344, b2-adrenoceptor agonists and selective b-adrenoceptor antagonists. Formation of 2-deoxy[1-14C]glucose-6-phosphate or 14CO2 was measured. 2-Deoxy[1-14C]-glucose uptake and b-adrenoceptor mRNA were measured in C2C12 cells. Key results: 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33–54%. The effect of BRL37344 was prevented by 1 mM atenolol but not by 300 nM CGP20712A or ICI118551, or 1 mM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 stimulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only b2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2deoxyglucose uptake and the effect of clenbuterol was not significant. Conclusions and implications: Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via b2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake. British Journal of Pharmacology (2008) 155, 395–406; doi:10.1038/bjp.2008.244; published online 16 June 2008 Keywords: b-adrenoceptor; atypical b-adrenoceptor; BRL37344; clenbuterol; salbutamol; soleus muscle; glucose uptake; C2C12 cells; fatty acid oxidation; ligand-directed signalling Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; dNTP, deoxynucleoside triphosphate; RT, reverse transcriptase Introduction b-Adrenoceptor agonists affect blood glucose homoeostasis in rodents by multiple mechanisms. Repeated administration of b3-adrenoceptor agonists improves insulin sensitivity in insulin-resistant rodents, perhaps because they increase fatty Correspondence: Professor JRS Arch, Clore Laboratory, University of Buckingham, Buckingham MK18 1EG, UK. E-mail: 1 Current address: Department of Molecular Medicine, School of Medical Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. 2 Current address: AstraZeneca, Radnor, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. 3 Current address: Division of Biology & Biomedical Sciences, Washington University, 660 South Euclid Avenue, St Louis, MO 63110, USA. Received 12 March 2008; revised 13 May 2008; accepted 20 May 2008; published online 16 June 2008 acid oxidation and lower intracellular lipid metabolite concentrations (Arch, 2002; Darimont et al., 2004). Single doses of b3-adrenoceptor agonists, in contrast, lower blood glucose in insulin-sensitive but not in insulin-resistant rodents. This effect is a consequence of increased insulin secretion, which is, in turn, probably a consequence of increased lipolysis and stimulation of insulin secretion by non-esterified fatty acids (Grujic et al., 1997). Single doses of b2-adrenoceptor agonists raise blood glucose levels by stimulating hepatic glucose output (Smith et al., 1990), possibly because they stimulate glucagon secretion (Lacey et al., 1991; Yoshida et al., 1991). b-Adrenoceptor agonists also affect glucose homoeostasis acutely, through direct effects on both brown adipocytes (Chernogubova et al., 2005) and skeletal muscle—the subject 396 b-adrenoceptor agonists and muscle metabolism RA Ngala et al of the present work. Very low concentrations of the selective b3-adrenoceptor agonist, BRL37344 (RR þ SS-(±)-4-(2-[(2-(3chlorophenyl)-2-hydroxyethyl)amino]propyl)phenoxyacetic acid) stimulated 2-deoxyglucose uptake by rat-isolated soleus and extensor digitorum longus muscle in vitro (Abe et al., 1993; Liu et al., 1996a). This effect was not blocked by atenolol (b1-adrenoceptor antagonist), ICI118551 ((±)-1[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol hydrochloride) (b2-adrenoceptor antagonist) or (except at a high concentration) by SR59230A (3-(2ethylphenoxy)-1[(1s)-1,2,3,4-tetrahydronaphth-1-ylamino]2S-propanol oxalate) (b3-adrenoceptor antagonist) (Liu et al., 1996a, b). Moreover, BRL37344 had its maximal effect at concentrations (10–100 pM) below its EC50 concentration (2 nM) for stimulation of lipolysis via rodent b3-adrenoceptors and far below concentrations that stimulate responses mediated by rodent b1- or b2-adrenoceptors (Arch et al., 1984). Very low concentrations (10–100 pM) also stimulated [1-14C]-palmitate and [2-14C]-pyruvate oxidation in soleus muscle from both wild-type (Board et al., 2000a) and, according to a preliminary report, b3-adrenoceptor null mice (Board et al., 2000b). It is also intriguing that higher concentrations of BRL37344, acting via what appeared to be a b2-adrenoceptor (as the effect was blocked by ICI118551 but not atenolol), inhibited rather than stimulated 2deoxyglucose uptake in rat soleus muscle (Liu et al., 1996a). b-Adrenoceptors that mediate effects of BRL37344 and other agonists on 2-deoxyglucose uptake in L6 rat skeletal muscle cells have also been characterized. No effects of very low concentrations of BRL37344 were reported, however, and, in contrast to rat skeletal muscle, higher concentrations stimulated 2-deoxyglucose uptake (Tanishita et al., 1997; Nevzorova et al., 2002, 2006). Nevertheless, as in rat soleus muscle, this effect appeared to be mediated by the b2adrenoceptor (Nevzorova et al., 2002). Similarly, isoprenaline-stimulated glycogen synthesis in L6 cells displayed a typical concentration–response curve, a typical EC50 value of 25 nM and sensitivity to antagonism by ICI118551 (Yamamoto et al., 2007). There are a number of examples of b-adrenoceptors displaying unusual pharmacology (Baker et al., 2003a; Arch, 2004; Baker, 2005a, b); it may be that the rat soleus muscle provides another example of this phenomenon. To investigate this (...truncated)