Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism - PubMed (original) (raw)
. 2012 Jul;32(14):2871-9.
doi: 10.1128/MCB.06575-11. Epub 2012 May 14.
Ben Murray, Sabine Gutzwiller, Stefan Marcaletti, David Marcellin, Sebastian Bergling, Sophie Brachat, Elke Persohn, Eliane Pierrel, Florian Bombard, Shinji Hatakeyama, Anne-Ulrike Trendelenburg, Frederic Morvan, Brian Richardson, David J Glass, Estelle Lach-Trifilieff, Jerome N Feige
Affiliations
- PMID: 22586266
- PMCID: PMC3416189
- DOI: 10.1128/MCB.06575-11
Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism
Brigitte Fournier et al. Mol Cell Biol. 2012 Jul.
Abstract
Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.
Figures
Fig 1
The activin receptor IIB inhibits the differentiation of primary brown adipocytes. (A to C) Primary brown preadipocytes isolated from mouse interscapular BAT were differentiated for 9 days using a classical adipogenic medium (AM) containing insulin and thyroid hormone for the entire protocol and IBMX, dexamethasone, and indomethacin for the first 2 days. Myostatin (Mstn or M; 10 ng/ml), a monoclonal antibody against the activin receptor IIB (ActRIIB Ab; 10 μg/ml), or a combination of both were added to the adipogenic medium, and fresh medium and treatments were replaced every 2 days. The level of differentiation was evaluated by visualizing lipid droplet accumulation using phase-contrast light microscopy (A) and by a nonbiased clustering analysis of expression microarray data from 2 independent cultures for each condition (B). The heat map in panel B is colorized according to gene expression from dark blue (low expression) to dark red (high expression). The average expression of all genes in clusters 1a, 1b, and 2 is represented below the heat map. (C) The expression of classical brown fat markers was measured by quantitative reverse transcription-PCR. (D) The supernatants from the primary brown adipocytes treated with control medium (CM) and adipogenic medium were subjected to a Smad2/3 reporter gene assay where a (CAGA)12-luciferase reporter was stably transfected into HEK293 T cells. The ActRIIB-dependent response was determined by adding an ActRIIB-Fc at 10 μg/ml. R.L.U., relative luciferase units.
Fig 2
ActRIIB inhibition in mice increases the amount of brown but not white fat. (A) SCID mice (n = 12/group) were treated for 4 weeks with a weekly subcutaneous injection of control (Ctrl) antibody (IgG1; 20 mg/kg/week) or increasing amounts of a human monoclonal antibody against ActRIIB (2, 6, and 20 mg/kg/week). The wet mass of gastrocnemius muscle, interscapular BAT, and epididymal white adipose tissue (WAT) was measured and expressed as the percent change from the average of the control group. *, P < 0.05. (B and C) Interscapular brown adipose tissue from control and ActRIIB Ab-treated mice (20 mg/kg/week) was imaged by light microscopy after laminin staining, and cell size was quantified by histomorphometry. (D) Epididymal white adipose tissue from control and ActRIIB Ab-treated mice (20 mg/kg/week) was imaged by light microscopy after hematoxylin-eosin staining. (E) Inguinal white adipose tissue from control and ActRIIB Ab-treated mice (20 mg/kg/week) was imaged by light microscopy after UCP-1 immunohistochemistry. Bars, 50 μm. (F) Quantitative reverse transcription-PCR measurement of relative UCP-1 levels in epididymal and inguinal white adipose tissue from control and ActRIIB Ab-treated mice (20 mg/kg/week).
Fig 3
ActRIIB inhibition increases lipid but not mitochondrial density. (A) Lipid droplet size was analyzed on hematoxylin-eosin-stained sections from interscapular BAT using automated detection and quantification software (n = 5/group). *, P < 0.05. (B) Mitochondrial DNA was quantified after DNA extraction by amplifying the mitochondrial gene cytochrome b by TaqMan quantitative PCR and normalizing to the levels of two probes detecting genomic DNA. (C) Interscapular brown adipose tissue from control and ActRIIB Ab-treated mice (20 mg/kg/week) was imaged by electron microscopy (C). L, lipid droplet; e, erythrocyte; N, nucleus, m, mitochondrion. Bars, 0.5 μm.
Fig 4
ActRIIB inhibition blocks Smad3 phosphorylation in BAT and activates oxidative metabolism. (A) Total and phosphorylated Smad3 protein levels in interscapular BAT of WT mice treated with IgG1 or the ActRIIB antibody at 20 mg/kg/week were measured by Western blotting. The quantification of two independent experiments is shown on the right. (B) Gene set enrichment analysis following microarray analysis of interscapular BAT of mice treated with IgG1 or the ActRIIB antibody at 20 mg/kg/week. Expression values were ranked and colorized from dark blue for low expression to dark red for high expression. Tg, transgenic. (C) Primary brown adipocytes were treated with myostatin (Mstn; 10 ng/ml), the ActRIIB Ab (10 μg/ml), or a combination of both, and gene expression was measured by quantitative reverse transcription-PCR using specific TaqMan probes for the indicated genes. *, P < 0.05.
Fig 5
The ActRIIB antibody does not potentiate BMP signaling. (A) Smad1/5/8 activity was measured using a BMP response element-luciferase reporter stably expressed in C28a2 cells, and Smad2/3 activity was measured with a (CAGA)12-luciferase reporter stably expressed in HEK293 cells. BMP-2, -4, and -7 were incubated at 200, 100, and 200 ng/ml, respectively, myostatin was incubated at 100 ng/ml, and the ActRIIB antibody was incubated at 10 μg/ml. PBS, phosphate-buffered saline. (B) Primary brown adipocytes were treated with BMP-7 (3 nM), the ActRIIB Ab (10 μg/ml), or a combination of both, as described in the legend to Fig. 1. *, P < 0.05; NS, not significant.
Fig 6
ActRIIB inhibition increases cold tolerance and energy expenditure via nonshivering thermogenesis. (A) Cold tolerance was evaluated by placing C57BL6/J mice (n = 11/group) that had been treated for 3 weeks with a weekly subcutaneous injection of vehicle or a mouse monoclonal antibody against ActRIIB (20 mg/kg/week) at 10°C for 4 h and measuring rectal body temperature every hour. (B and C) Energy expenditure was measured for 20 to 24 h by indirect calorimetry for mice treated as described for panel A for 4 weeks. Measurements were performed at 22°C, 10°C (cold), or 30°C (thermoneutrality) for independent groups of mice (n = 8 to 11/group). Data were integrated over the dark (B) or the light (C) phase to maximize the effects of temperature. (D) The oxygen consumption rate in primary brown adipocytes treated with myostatin (Mstn; 10 ng/ml) or the ActRIIB Ab (10 μg/ml) (n = 10 per condition) was measured under basal conditions or in the presence of oligomycin (uncoupled) or FCCP (maximal). *, P < 0.05.
References
- Calvo JA, et al. 2008. Muscle-specific expression of PPARgamma coactivator-1alpha improves exercise performance and increases peak oxygen uptake. J. Appl. Physiol. 104:1304–1312 - PubMed
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