The β3-adrenergic receptor agonist mirabegron improves glucose homeostasis in obese humans

Abstract

BACKGROUNDBeige adipose tissue is associated with improved glucose homeostasis in mice. Adipose tissue contains β3-adrenergic receptors (β3-ARs), and this study was intended to determine whether the treatment of obese, insulin-resistant humans with the β3-AR agonist mirabegron, which stimulates beige adipose formation in subcutaneous white adipose tissue (SC WAT), would induce other beneficial changes in fat and muscle and improve metabolic homeostasis.METHODSBefore and after β3-AR agonist treatment, oral glucose tolerance tests and euglycemic clamps were performed, and histochemical analysis and gene expression profiling were performed on fat and muscle biopsies. PET-CT scans quantified brown adipose tissue volume and activity, and we conducted in vitro studies with primary cultures of differentiated human adipocytes and muscle.RESULTSThe clinical effects of mirabegron treatment included improved oral glucose tolerance (P < 0.01), reduced hemoglobin A1c levels (P = 0.01), and improved insulin sensitivity (P = 0.03) and β cell function (P = 0.01). In SC WAT, mirabegron treatment stimulated lipolysis, reduced fibrotic gene expression, and increased alternatively activated macrophages. Subjects with the most SC WAT beiging showed the greatest improvement in β cell function. In skeletal muscle, mirabegron reduced triglycerides, increased the expression of PPARγ coactivator 1 α (PGC1A) (P < 0.05), and increased type I fibers (P < 0.01). Conditioned media from adipocytes treated with mirabegron stimulated muscle fiber PGC1A expression in vitro (P < 0.001).CONCLUSIONMirabegron treatment substantially improved multiple measures of glucose homeostasis in obese, insulin-resistant humans. Since β cells and skeletal muscle do not express β3-ARs, these data suggest that the beiging of SC WAT by mirabegron reduces adipose tissue dysfunction, which enhances muscle oxidative capacity and improves β cell function.TRIAL REGISTRATIONClinicaltrials.gov NCT02919176.FUNDINGNIH: DK112282, P30GM127211, DK 71349, and Clinical and Translational science Awards (CTSA) grant UL1TR001998.

Keywords: Adipose tissue; Clinical Trials; Glucose metabolism; Metabolism; Obesity.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Flow chart of the study design and analysis of the research participants.

Sixty-seven research subjects were assessed, and 39 were randomized into 3 drug treatment groups. The results for the subjects in the pioglitazone and combination therapy groups will be presented in a future publication. Thirteen subjects were in the mirabegron treatment group, and all completed the study.

Figure 2. Mirabegron treatment improves glucose homeostasis in obese, insulin-resistant subjects.

Subjects were treated with mirabegron (50 mg/day) for 12 weeks. OGTTs and euglycemic clamps were performed at baseline and after treatment. (A) OGTT results and AUC for all subjects (n = 12). Data indicate the mean ± SEM. OGTT data were analyzed by a 2-way, repeated-measures ANOVA (***P < 0.001 and ****P < 0.0001); AUC data were analyzed by a paired, 2-tailed Student’s t test (**P < 0.01). (B) Euglycemic clamping was performed at an insulin infusion rate of 1.0 mU/kg/min, and the GIR was determined before and after treatment. Data indicate the mean ± SEM (n = 13) and were analyzed by a paired, 2-tailed Student’s t test (*P < 0.05). (C) The insulinogenic index was determined from the results of the oral glucose tolerance test. Data indicate the mean ± SEM (n = 12) and were analyzed by a paired, 2-tailed Student’s t test (*P < 0.05). (D) The disposition index is the product of (C) insulin sensitivity and (D) the insulinogenic index. Data indicate the mean ± SEM (n = 12). **P < 0.01, by paired, 2-tailed Student’s t test. (E) BAT volume was quantified by PET-CT scans before and after treatment. Eight subjects had no BAT at baseline, and BAT did not increase after treatment of these subjects. (F) The change in SC WAT beiging was calculated as the difference in UCP1 protein expression before and after treatment. The change in the disposition index (D) and the change in UCP1 in SC WAT were analyzed by regression analysis. The Spearman’s correlation coefficient and P value are indicated in F.

Figure 3. Mirabegron treatment reduces skeletal muscle TGs, but not toxic lipids, and promotes fiber-type switching to type I fibers.

Lipids were extracted from skeletal muscle obtained from vastus lateralis biopsies and the levels of the indicated lipids were measured as described in Methods. The levels of (A) TG, (B) DAG, and (C) ceramide before and after mirabegron treatment were determined. (D–G) mRNA expression of genes in muscle was determined by real-time RT-PCR. (H) Representative images of muscle stained for MyHC I, MyHC IIa, and MyHC IIx before and after mirabegron treatment. Scale bars: 50 μm. (I–L) Quantification of type I, type IIa, and type IIx fibers. Data are expressed as the percentage of total fibers and indicate the mean ± SEM (n = 12–13). *P < 0.05 and **P < 0.01, by paired, 2-tailed Student’s t test.

Figure 4. CM isolated from adipocytes treated with mirabegron induces PGC1α expression in human myotubes in vitro.

Differentiated human adipocytes, with or without 100 nM mirabegron treatment for 16 hours, and the CM was isolated as described in Methods. An additional control was made by adding mirabegron to the CM after the CM was isolated from the adipocytes. The human myotubes were then incubated with 0.025% DMSO (Control), mirabegron (25 nM), adipocyte CM (25%), mirabegron-treated adipocyte CM (25%), or adipocyte CM plus mirabegron (25%). The final concentration of mirabegron was 25 nM. Data indicate the mean ± SEM (n = 3). *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Tukey’s multiple comparisons test.

Figure 5. Mirabegron treatment stimulates lipolysis.

(A) Adipose tissue (0.5 g) from the SC WAT biopsy was placed in medium and kept at 37°C for 1 hour, and the level of glycerol was determined in the adipose tissue explant CM before and after treatment. (B) Plasma NEFA levels were determined before and after treatment. Data indicate the mean ± SEM (n = 13). **P < 0.01 and ***P < 0.001, by paired, 2-tailed Student’s t test.

Figure 6. Mirabegron treatment increases alternatively activated macrophages in SC WAT.

To characterize macrophage polarization, adipose tissue sections were doubly stained for (A) CD86 and CD68 (M1) and (B) CD163 and CD68 (M2). (C) UCP1 was present in CD163+ cells in SC WAT. Yellow arrows point to UCP1+CD163+DAPI+ cells (scale bar: 50 μm). (D) UCP1+CD163+ cells were quantified in SC WAT before and after mirabegron treatment. Data indicate the mean ± SEM (n = 13). *P < 0.05 and ***P < 0.001, by paired, 2-tailed Student’s t test.

Figure 7. Mirabegron treatment reduces state 4 respiration but does not increase uncoupled respiration in purified mitochondria isolated from SC WAT.

Mitochondria were purified and the bioenergetics analyzed using an Oxytherm before and after mirabegron treatment as described in Methods. This involved the sequential addition of adenosine diphosphate (ADP), oligomycin (Oligo), FFA (60 μM linoleic acid), fatty acid–free BSA, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) (10 μm). (A and B) OCRs before and after treatment are shown. (C) The difference between oligomycin and BSA was calculated before and after treatment. Data indicate the mean ± SEM (n = 12). *P = 0.05, by Wilcoxon matched-pairs, signed-rank test.