The metabolic effects of mirabegron are mediated primarily by β3‐adrenoceptors

Abstract The β3‐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, [3H]‐2‐deoxyglucose uptake, cellular glycolysis, and O2 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 β3‐adrenoceptors as they were largely absent in adipocytes derived from β3‐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 β3‐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 β3‐adrenoceptor.


| INTRODUC TI ON
The β 3 -adrenoceptor was for many years a target in pharmaceutical drug programs for the development of antiobesity or antidiabetic drugs due to its high expression in rodent adipocytes, where its activation results in increased lipolysis, thermogenesis, and weight loss. 1,2 Most screening was done in rodent models of obesity leading to the development of what turned out to be rodent-selective β 3 -adrenoceptor agonists, such as CL316243. 3 Although these compounds were active in in vitro cell-based assays expressing high levels of human β 3 -adrenoceptor, they had little effect at physiological levels of expression 4,5 leading to failure of the compounds in phase 2 clinical trials for the treatment of obesity or diabetes. The major factors involved were lack of efficacy of β 3 -adrenoceptor agonists at the human receptor and poor bioavailability. 6 However, β 3 -adrenoceptors have re-emerged as a target for metabolic disease in the past 10 years since the identification of functional brown adipose tissue (BAT) in adult humans 7 and the discovery of brite (brown within white) adipocytes. 8 Brite adipocytes (or beige adipocytes) are brown-like adipocytes located predominately in white adipose tissue (WAT) depots, and the process of browning has been advocated as a means to treat obesity and metabolic disease. 8 Stimulation of β 3 -adrenoceptors in rodent brite adipocytes causes increased oxygen consumption by activation of UCP1 and increased glucose uptake, 9,10 thus identifying a novel mechanism for the development of antiobesity and antidiabetic therapeutics.
Recently, a human active β 3 -adrenoceptor agonist, mirabegron (Astellas Pharma Inc), was approved for the treatment of overactive bladder syndrome, but also has potential to be used for obesity/metabolic disease. 11 Although there is limited information on the metabolic effects of mirabegron, one recent study showed that chronic treatment of mice fed a high-fat diet lowered body weight, fat mass, and improved glucose tolerance. 12 In lean humans, acute mirabegron administration positively affected BAT activity and resting metabolic rate, 13,14 whereas in obese humans, chronic mirabegron treatment led to improved glucose tolerance and insulin sensitivity. 15 However, while mirabegron is recognized as a β 3 -adrenoceptor agonist, its selectivity has been questioned, and it does have off-target actions. 11 These include actions at α 1 -and β 1 -adrenoceptors, muscarinic receptors, noradrenaline and dopamine transporters, organic cation transporters, p-glycoprotein, and 2 cytochrome P450 enzymes.
This study therefore investigates the effects of mirabegron in brown, white, and brite adipocytes in vitro, and its effects on glucose utilization and thermogenesis in vivo. We have utilized β 3 -and β 1/2 -adrenoceptor knockout mice to assess selectivity.
Additionally, we have performed glucose tolerance tests in UCP1 knockout mice to address the role of this protein in mediating the acute effects of mirabegron on blood glucose levels in vivo.
Rosiglitazone was obtained from Tocris Bioscience. All other reagents were of analytical quality.

| Animals
All experiments complied with the Australian code for the care Maintenance diet for mice, 24% protein, 15% fat, and 61% carbohydrate) and water. FVB/N mice were the wild-type control for the β 3 -adrenoceptor knockout 16 mice, DBAxC57 mice were the wild-type control for the β 1/2 -adrenoceptor knockout 17 mice, and Bl6/129sv mice were the wild-type control for the UCP1 knockout mice. A summary of the number of mice used for each experiment is given in

| CHO cell culture
Chinese Hamster ovary (CHO) cells stably expressing either the human 19 or mouse 20 β 3 -adrenoceptor, the human β 1 -adrenoceptor, or the human β 2 -adrenoceptor were grown in 50:50 DMEM/Ham's F12 medium supplemented with 5% (vol/vol) fetal bovine serum (FBS) and 2-mM L-glutamine at 37°C in 5% CO 2 . After plating, cells were maintained in growth medium overnight before being serum starved for ~16 h prior to experimentation. Cells were routinely tested for mycoplasma.

| L6 cell culture
L6 cells over expressing GLUT4 with a myc epitope (kindly provided by Amira Klip, Hospital for Sick Children, Toronto, Canada) were grown in DMEM supplemented with 4-mM L-glutamine, 10% (vol/vol) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10-µM HEPES at 37°C in 5% CO 2 . Cells were grown as myoblasts by ensuring that cells were kept at less than 70% confluency. Upon confluency (90%), differentiation was induced by lowering the FBS concentration to 2% (vol/vol) for 7 days, with media changed every 2 days.

| Measurement of cyclic AMP levels
Adipocytes were grown in 96-well plates for 7 days before being serum starved in DMEM/Hams F12 media containing 0.5% (w/vol) BSA, 2.4-nM insulin, 25 µg/mL sodium ascorbate, 10-mM HEPES, 4-mM L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin, with 1-µM rosiglitazone for brite adipocytes only. CHO and L6 cells were grown in 96-well plates. cAMP assays were performed as described previously 10 and cAMP content was measured (αScreen kits; PerkinElmer Life Sciences) according to the manufacturer's instructions. Forskolin (100 µM; this concentration produced ~16-fold increase in cAMP levels) was used as positive control in all cAMP assays to ensure an intact Gs-cAMP pathway (data not shown).

| Reverse transcription-qPCR
Adipocytes were grown in 6-well plates for 7 days and were serum starved as described above. In the morning, media were replaced by DMEM containing 25-mM glucose, 0.5% (w/vol) BSA, and 0.125-mM sodium ascorbate for 30 min, before mirabegron (1 µM Adipocyte cultures a : Cyclic AMP 18 10 In vivo glucose uptake 9 12 In vivo oxygen consumption 21 13 Glucose tolerance test

| Measurement of oxygen consumption and extracellular acidification rates
Oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) were measured using the Seahorse xF96 (Seahorse Bioscience). Adipocytes were grown in Seahorse cell culture plates as above. On day 7, adipocytes were washed twice in XF assay medium (Seahorse Bioscience) supplemented with 25-mM glucose, 0.5-mM sodium pyruvate, 2-mM L-glutamine and 1% (w/vol) fatty acid-free BSA, and 180-µL added/well. OCR and ECAR were measured 21 with some modifications. 10 Six baseline rate measurements were made using a 2 min mix, 5 min measure cycle. Agonists (20 µL) were injected pneumatically by the machine into each well, mixed, and 10 measurements were made using the 2 min mix, 5 min measure cycle. OCR and ECAR rates immediately prior to compound injection were used as the basal rates and defined as 100%.

| Whole body oxygen consumption
Oxygen consumption was measured to determine non-shivering thermogenic capacity following mirabegron injection using a Somedic INCA apparatus, principally as described previously. 24 FVB/N and β 3 -adrenoceptor knockout mice (12-week-old male mice) were anesthetized (85 mg/kg i.p. pentobarbital sodium) and placed in the chambers set to 30°C for ~25 min to estimate the basal metabolic rate. Thereafter, the mice were removed from the chamber briefly, injected with mirabegron (1 mg/kg i.p., 1.2% ethanol in saline) or vehicle (1.2% ethanol in saline), and placed back in the chambers for further measurements. At the completion of the experiment, mice were killed by CO 2 inhalation and cervical dislocation.

| In vivo glucose uptake
Mice ( were dissected and lysed in 0.5 M NaOH at 60°C for at least 2 hours, and radioactivity was measured by liquid scintillation counting.

| Statistical Analysis
All data are expressed as mean ± SEM of n. For concentrationresponse data, curves were analyzed using nonlinear curve fitting

Mirabegron increased cAMP levels in all CHO-K1 cells express-
ing β-adrenoceptor subtypes, but was a weak partial agonist at human β 1 -and β 2 -adrenoceptors compared with the prototypical β-adrenoceptor agonist isoprenaline. It was ~2800 and ~1380 selective for the human β 3 -compared to β 1 -or β 2 -adrenoceptors, consistent with previous results. 4 It was a full agonist at the human and the mouse β 3 -adrenoceptors. Thus, at cloned receptors, mirabegron is a potent and β 3 -selective adrenoceptor agonist.

| Mirabegron increases β 3 -adrenoceptormediated glucose clearance in BAT and inguinal WAT
To determine the effects of acute mirabegron administration on glucose tolerance and whether this was mediated through  Figure 2E).
In vivo glucose uptake assays were performed to gauge the contribution of metabolically active tissues toward improvements in glucose clearance observed in mirabegron-treated mice.
Mirabegron treatment was associated with a robust increase in 3 H-2-deoxyglucose uptake into BAT ( Figure 2G Table 2).
To supplement in vivo findings, primary brown adipocytes were derived from FVB/N, DBAxC57, β 1/2 -adrenoceptor, or β 3 -adrenoceptor knockout mice and in vitro glucose uptake assays were performed. In cells from FVB/N, β 1/2 -adrenoceptor knockout, and DBAxC57 mice, insulin, isoprenaline, and mirabegron all increased cellular glucose uptake in a concentration-dependent manner (Table 3; Figure 2K,M,N). In primary cell cultures of brown adipocytes from β 3 -adrenoceptor knockout mice, insulin and isoprenaline stimulation caused marked increases in glucose uptake, whereas mirabegron had no effect, highlighting the essential role of the β 3 -adrenoceptor in the actions of mirabegron (Table 3; Figure 2L).
Mirabegron also increased glucose uptake in brown adipocytes derived from FVB/N mice pretreated with a β 1 -or β 2 -antagonist but failed to do so after treatment with a β 3 -adrenoceptor antagonist ( Figure 3A,B). These results strongly suggest that the effects of mirabegron on glucose uptake in brown adipocytes are mediated through the β 3 -adrenoceptor. Taken together, the results suggest that acute administration of mirabegron markedly improves glucose tolerance by increasing glucose uptake into BAT and inguinal WAT following β 3 -adrenoceptor activation.

| Mirabegron administration causes β 3 -adrenoceptor-mediated thermogenic activation of BAT
cAMP production is a major determinant of BAT thermogenic activity. 2 To examine whether mirabegron increased cAMP levels in these cells, primary brown adipocytes were isolated from FVB/N or β 3 -adrenoceptor knockout mice and treated with isoprenaline or mirabegron. In primary brown adipocytes derived from FVB/N mice, mirabegron and isoprenaline increased cAMP levels in a concentration-dependent manner, whereas responses to mirabegron but not isoprenaline were attenuated in adipocytes derived from β 3adrenoceptor knockout mice (Table 4; Figure 4A,B).
To determine whether increases in cAMP production following mirabegron in vitro translated into increased thermogenic activity, OCR was measured in primary brown adipocytes using plate-based respi- TA B L E 2 Ability of mirabegron to increase cAMP levels in cells expressing different β-adrenoceptor subtypes adipocytes derived from FVB/N mice ( Figure 4C). In brown adipocytes derived from β 3 -adrenoceptor knockout mice, the effects of isoprenaline were still evident most likely due to its effects on β 1 -adrenoceptors that can compensate for the lack of β 3 -adrenoceptors in these cells. 23 Interestingly, mirabegron was still able to increase OCR in brown adipocytes derived from β 3 -adrenoceptor knockout mice ( Figure 4D), albeit this response was modest compared to that in brown adipocytes derived from FVB/N mice.
ECAR was measured in tandem using the Seahorse xF96 analyzer as a proxy for glycolytic activity in these cells where pyruvate is reduced to lactate. In adipocytes derived from FVB/N mice, isoprenaline and mirabegron treatment increased ECAR several fold in brown adipocytes ( Figure 4E). Noticeably, isoprenaline and mirabegron still increased ECAR in brown adipocytes derived from β 3 -adrenoceptor knockout mice, although the effect was much smaller than in brown adipocytes derived from FVB/N mice, suggesting a non-β 3 -adrenoceptor-mediated effect of mirabegron and isoprenaline on glycolytic activity in these cells ( Figure 4F).
Additionally, to investigate whether mirabegron could directly recruit BAT, UCP1 expression was measured in primary brown adipocytes from FVB/N mice where treatment significantly increased UCP1 mRNA. This effect was absent in brown adipocytes from β 3 -adrenoceptor knockout mice ( Figure 4G).

Finally, to determine if mirabegron-mediated increases in cAMP
and OCR were sufficient to increase whole body energy expenditure in vivo, indirect calorimetry was performed where FVB/N or β 3 -adrenoceptor knockout mice were injected with vehicle or mirabegron ( Figure 4H,I). Oxygen consumption was increased significantly by mirabegron in FVB/N but not in β 3 -adrenoceptor knockout mice, showing that mirabegron recruits and activates BAT in a β 3 -adrenoceptor-dependent manner.

| Mirabegron promotes browning and increases β 3 -adrenoceptor-dependent thermogenic activity in brite adipocytes
Chronic mirabegron treatment is associated with browning of subcutaneous WAT in humans 15 and inguinal WAT in rodents. 12 To address the importance of the β 3 -adrenoceptor for browning and thermogenic activation of these cells, white adipocytes derived from FVB/N or β 3 -adrenoceptor knockout mice were treated with rosiglitazone for 7-8 days to acquire a brite phenotype, that displayed upregulated levels of UCP1 expression relative to white adipocytes 9,10,26 ( Figure 5A).

In brite adipocytes, mirabegron treatment markedly increased UCP1
expression. In the absence of the β 3 -adrenoceptor, as observed in brown adipocytes, mirabegron had no effect on UCP1 expression in white or brite adipocytes ( Figure 5B). TA B L E 3 Ability of mirabegron, isoprenaline, and insulin to increase glucose uptake in brown, brite, and white adipocytes The effect of mirabegron and isoprenaline on cAMP, OCR, ECAR, and glucose uptake was measured in white and brite adipocytes derived from FVB/N or β 3 -adrenoceptor knockout mice.

| Mirabegron-mediated improvements in glucose tolerance are UCP1-dependent but insulinindependent in vivo
Our results show that mirabegron activates thermogenesis and increases glucose uptake into brown and brite adipocytes, and improves glucose tolerance in vivo highlighting the feasibility of β 3adrenoceptors as a target in metabolic disease. To address whether UCP1 is required for the observed improvements in glucose tolerance with mirabegron, UCP1 knockout and wild-type controls were treated with mirabegron 30 minutes prior to glucose challenge.
Mirabegron treatment produced significant improvements in glucose clearance in wild-type mice but had no effect in UCP1 knockout animals ( Figure 6A-C).
Given previous findings that mirabegron has off-target activity, 11 we confirmed that the improved glucose tolerance was not due to a direct effect on insulin release from pancreatic islets. Incubation of islets in high glucose conditions (16.7 mM) increased insulin secretion compared to low glucose conditions (2.8 mM) ( Figure 6D).

Forskolin increased glucose-stimulated insulin secretion in mouse
islets compared to islets incubated with 16.7-mM glucose alone ( Figure 6D). Mirabegron did not have an additional effect on insulin secretion compared with insulin secreted from islets treated with 16.7-mM glucose alone ( Figure 6D). Since mirabegron does not directly affect insulin release from the pancreas, the acute effects of mirabegron on blood glucose clearance in vivo are therefore fully dependent on its effects on UCP1.

| D ISCUSS I ON
Although the main role for BAT is thermogenesis, 2 it also represents an important glucose-clearing organ displaying significant glucose uptake per gram of tissue, that in cold-exposed obese mice contributes ~75% of whole body glucose uptake. 27 In chow and high-fat diet-fed mice, BAT transplantation decreases body weight, increases glucose metabolism and insulin sensitivity, and increases glucose uptake into BAT and WAT. 28 This makes BAT an attractive target for treating hyperglycemia associated with metabolic disease. We show here that mirabegron increases glucose uptake and glycolysis in mouse brown adipocytes in vitro and promotes glucose uptake into BAT in vivo. This results in rapid large improvements in glucose tolerance, likely attributable to increased glucose uptake into BAT, rather than skeletal muscle, another major site for glucose disposal. It has been suggested that β 3 -adrenoceptor-mediated glucose uptake into

BAT occurs indirectly through an insulin-dependent mechanism,
whereby β 3 -adrenoceptor-mediated lipolysis leads to pancreatic insulin release, that then increases glucose uptake into BAT. 29,30 Our results do not support the involvement of insulin since mirabegron in vivo would then be expected to also increase glucose uptake into skeletal muscle, an effect that was not observed. Additionally, we observed no direct actions of mirabegron on glucose-stimulated insulin secretion from isolated pancreatic islets.
While mirabegron was developed as a human β 3 -adrenoceptor agonist, it has off-target actions on heart rate that could be due to actions at cardiac β-adrenoceptors, 11 but may also be indirect due to release of noradrenaline. 31 In one study of the metabolic effects of mirabegron in humans where large doses (four times the usual therapeutic dose) were given, unwanted increases in heart rate and systolic blood pressure were all too evident. 14 Here, we examined the β-adrenoceptor selectivity of mirabegron in CHO cells expressing human β-adrenoceptor subtypes, in cultured mouse adipocytes and in vivo using mice with genetic deletion of either the β 3 -adrenoceptor or combined β 1/2 -adrenoceptor. All responses to mirabegron were lost or substantially depleted in the β 3 -adrenoceptor knockout mice, but unaffected in the β 1/2 -adrenoceptor knockout animals. The small but significant residual cAMP, OCR, and ECAR responses to mirabegron occurred only in brown adipocytes derived from β 3 -adrenoceptor knockout mice. It has been shown previously that mirabegron acts as a partial agonist at rat β 1 -adrenoceptors (intrinsic activity 0.6 relative to isoprenaline, pEC 50 6.21), despite having negligible direct activity at the human β 1 -adrenoceptor, and at β 2 -adrenoceptors from both species. 4,32 Since cultured mouse brown adipocytes express β 1 -adrenoceptor mRNA, 10 it is likely that the residual responses to mirabegron in β 3 -adrenoceptor knockout mice are mediated by the F I G U R E 4 Mirabegron increases cAMP levels, OCR and ECAR rates, and UCP1 mRNA levels in brown adipocytes via activation of β 3 -adrenoceptors. Cyclic AMP accumulation in response to isoprenaline or mirabegron in adipocytes derived from (A) FVB/N (n = 5) or (B) β 3 -adrenoceptor knockout (n = 7) mice. (C, D) Effect of acute addition of isoprenaline (1 µM) or mirabegron (1 µM) on OCR in brown adipocytes derived from FVB/N (n = 6 isoprenaline; n = 12 mirabegron) or β 3 -adrenoceptor knockout (n = 6 isoprenaline; n = 5 mirabegron) mice. (E, F) Effect of acute addition of isoprenaline (1 µM) or mirabegron (1 µM) on ECAR in brown adipocytes derived from FVB/N (n = 5 isoprenaline; n = 11 mirabegron) or β 3 -adrenoceptor knockout (n = 6 isoprenaline; n = 6 mirabegron) mice. (G) Effect of mirabegron (1 µM, 24 h) on Ucp1 mRNA expression in brown adipocytes derived from FVB/N (n = 6) or β 3 -adrenoceptor knockout (n = 5) mice. Data are mean ± SEM of 5-6 independent experiments performed in duplicate. Data analyzed using Student's paired t-test (***P < .001). (H) In FVB/N mice, mirabegron (1 mg/kg i.p.; n = 9) administration significantly increased O 2 consumption rates in FVB/N mice as compared to vehicletreated mice (n = 12). (I) In β 3 -adrenoceptor knockout mice, mirabegron (1 mg/kg i.p.; n = 7) administration had no effect on O 2 consumption rates as compared to vehicle-treated mice (n = 6). Data analyzed using a two-way ANOVA with a Bonferroni post hoc test (*P < .05) β 1 -adrenoceptor. Interestingly, a recent paper 33 suggested that the β 1 -and not the β 3 -adrenoceptor is the primary adrenergic regulator of human brown adipocyte metabolism. Using a human immortalized brown adipocyte cell model, they showed that mirabegron had no effect on lipolysis or on the expression of several genes including UCP1. 33 This correlated with little expression of β 3 -adrenoceptors Overall, mirabegron is a β 3 -selective adrenoceptor agonist at the human receptor, as previously shown using cloned receptors. 4 This contrasts with other agonists previously trialed for treatment of obesity and/or diabetes. One early study using BRL35135, an F I G U R E 6 UCP1 is required for improved in vivo glucose tolerance in response to mirabegron. (A) In UCP1+/+ mice, mirabegron (1 mg/ kg i.p.; n = 6) administration 30 min prior to a glucose challenge (2 g/kg i.p.) significantly reduced blood glucose levels at 20, 40, and 60 min compared with control-treated (20% DMSO in saline, i.p.; n = 4) UCP1+/+ mice. (B) In UCP1-/-mice, mirabegron (1 mg/kg i.p.; n = 5) administration 30 min prior to a glucose challenge (2 g/kg i.p.) had no effect on blood glucose levels compared with control-treated (20% DMSO in saline, i.p.; n = 5) UCP1-/-mice. Data analyzed using a two-way ANOVA with a Bonferroni post hoc test (**P < .01). (C) Area under the curve analysis of glucose levels from panels A and B shows that mirabegron (1 mg/kg i.p) significantly reduces glucose levels in UCP1+/+ but not in UCP1−/− mice. Data analyzed using unpaired t-test (**P < .001). (D) Mirabegron does not affect glucose-stimulated insulin secretion in isolated mice islets. Each bar represents mean ± SEM from three incubations of 3 islets/well performed in three separate experiments. Forskolin (1 µM) increases glucose-stimulated insulin secretion compared with untreated islets (***P < .001 compared with control) (one-way ANOVA with Tukey multiple comparison test). In contrast, mirabegron at all concentrations did not affect stimulated insulin compared with untreated islets esterified prodrug that is quickly converted to BRL37344 in vivo, showed that chronic (10 days) administration of BRL35135 in obese subjects improves glycemic control, reduces insulin resistance (improved glucose tolerance profiles), and increases insulin-mediated body glucose disposal under hyperinsulinemic-euglycemic clamps. 35 However, there is convincing evidence that these effects of BRL35135 and its active metabolite BRL37344 primarily result from direct actions at β 2 -adrenoceptors in skeletal muscle. 36,37 To examine whether mirabegron acts in vivo on β 1/2 -adrenoceptors, we performed glucose tolerance tests in β 1/2 -adrenoceptor knockout mice.
Mirabegron still lowered blood glucose levels in these mice, findings that together with the increases in glucose uptake in skeletal mice in wild-type mice suggest that the improved glucose-clearing actions of mirabegron in mice are due to activation of β 3 -adrenoceptors.
There is tremendous interest in the role of brite adipocytes, defined as brown-like adipocytes located predominately in WAT depots. These adipocytes differ from brown adipocytes in that they originate from a white adipocyte precursor cell, and are converted to brite adipocytes in vitro and in vivo by environmental stimuli such as cold exposure, or a range of pharmaceutical agents. 8 Brite adipocytes have been advocated as a target for the treatment of metabolic disease, including obesity and type 2 diabetes, and are linked to obesity resistance in several mouse models. [38][39][40] Here we used rosiglitazone, a PPARγ agonist, to convert white adipocytes to brite adipocytes in vitro, as shown previously. 10,26 There were minimal effects of mirabegron to increase OCR levels or glucose uptake in white adipocytes, consistent with the low expression of β 3 -adrenoceptors in these cells and their low levels of UCP1 and mitochondrial content. 10 However, in brite adipocytes, mirabegron increased cyclic AMP levels, UCP1 mRNA levels, UCP1-mediated oxygen consumption, glucose uptake, and glycolysis in vitro, consistent with our previous study using β 3 -adrenoceptor agonists, such as CL316243, or noradrenaline. 10 It has been proposed that human BAT is actually comprised of brite adipocytes, 41 and further studies have shown that depending on the location in the body, humans most likely possess both BAT and brite adipocytes. 42,43 One recent study 12 showed that mirabegron (2 mg/kg by osmotic mini-pumps) resulted in a reduction in body weight, fat mass, and improved glucose tolerance tests in mice fed a high-fat diet that was associated with the browning of inguinal WAT. In obese humans, administration of mirabegron at 50 mg per day for 12 weeks has been reported to increase BAT volume resulting in improvements in oral glucose tolerance and insulin resistance without weight loss. 15 In the same study, an increased browning of subcutaneous white fat was reported that was associated with improved insulin sensitivity suggesting that these effects could be due to brite adipose tissue expansion and the induction of both UCP1-dependent and -independent mechanisms. We have shown that mirabegron treatment results in significantly improved glucose clearance primarily through the activation of β 3 -adrenoceptors. The presence of UCP1 is essential for this effect at least in lean mice housed at sub-thermoneutral temperatures. It has been recently reported that UCP1 knockout mice exhibit lower glucose uptake in thermogenic tissues with compensatory increases in WAT and skeletal muscle in an oral glucose tolerance test. 44 Our results imply that these compensatory mechanisms, if further induced by mirabegron in UCP1 knockout mice, are unable to significantly affect postprandial glucose uptake in these tissues to result in substantial increases in glucose tolerance. Although UCP1 is absent in BAT in UCP1 knockout mice, and the acute effects of mirabegron on blood glucose are attenuated, chronic administration may still lead to some induction of UCP1 and possibly other UCP1-independent mechanisms in brite adipocytes. This notion is supported by the observation that chronic mirabegron treatment of obese subjects, who have very little BAT, still led to improvements in glucose tolerance and insulin resistance, 15 although no effects on weight loss were shown.
The lack of effect on weight loss was speculated 15 to be due to the relatively low levels of BAT in humans; the dose of mirabegron used in the study (50 mg/day); that this dose of mirabegron was insufficient for browning of WAT and activation of UCP1; or that humans compensate for increased energy expenditure by increased food intake.
There is also great interest in pharmacological/environmental stimuli that can convert white adipocytes to brite adipocytes, as a means to combat obesity. One limitation is that while a range of agents 8 can increase the appearance of brite adipocytes, these adipocytes do not become thermogenic until activated by β 3 -adrenoceptor activity. To increase UCP1 mRNA/protein levels, we treated white adipocytes with rosiglitazone (a PPARγ agonist) to produce brite adipocytes that could then be activated by β 3 -adrenoceptor ligands. 10,26 Advantages of pursuing mirabegron for the treatment of obesity/diabetes include that it is well tolerated with a high level of adherence, is orally bioavailable, and has already met requirements set out by regulatory bodies for use in humans.
Furthermore, there is a growing body of literature that suggests mirabegron administration results in a pronounced improvement in various homeostatic parameters associated with metabolic syndrome. 15,45

| Perspectives and Significance
In conclusion, our study shows that mirabegron is a β 3 -selective adrenoceptor agonist and has direct β 3 -adrenoceptor-mediated effects on adipocytes that include increases in cAMP, glucose uptake, ECAR, UCP1 mRNA, and OCR. This results in increased adipose tissue glucose uptake, whole body oxygen consumption, and improved glucose tolerance. Additionally, UCP1 is essential for the acute effect of mirabegron on glucose tolerance in vivo.
Most previous studies investigating β 3 -adrenoceptors in mouse adipose tissue/adipocytes have utilized the β-adrenoceptor agonist CL316243. While this is a potent and selective β 3 -adrenoceptor agonist in rodents, it has limited actions at even the cloned human β 3 -adrenoceptor, and has poor bioavailability, preventing its use in humans. Conversely, while mirabegron is a safe orally active β 3 -adrenoceptor agonist that is used clinically for overactive bladder, there is limited publically available information on its metabolic actions. This study demonstrates that mirabegron displays very similar properties to CL316243 with respect to cAMP, glucose uptake, ECAR, UCP1 mRNA, and OCR in brown, white, and brite adipocytes, and increased adipose tissue glucose uptake, whole body oxygen consumption, and improved glucose tolerance in vivo. Therefore, mirabegron displays many of the positive metabolic effects of CL316243, is a β 3 -selective adrenoceptor agonist, and is known to be active in humans suggesting that chronic administration may be useful in the treatment of obesity and diabetes.

DATA S H A R I N G A N D DATA ACCE SS I B I LIT Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.