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, 342 (1), 106-18

The β2-adrenoceptor Agonist Formoterol Stimulates Mitochondrial Biogenesis

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The β2-adrenoceptor Agonist Formoterol Stimulates Mitochondrial Biogenesis

Lauren P Wills et al. J Pharmacol Exp Ther.

Abstract

Mitochondrial dysfunction is a common mediator of disease and organ injury. Although recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. To examine the impact of the β-adrenoceptor (β-AR) signaling pathway on MB, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes were exposed for 24 h to multiple β-AR agonists: isoproterenol (nonselective β-AR agonist), (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL 37344) (selective β(3)-AR agonist), and formoterol (selective β(2)-AR agonist). The Seahorse Biosciences (North Billerica, MA) extracellular flux analyzer was used to quantify carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterenol and BRL 37244 did not alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the β-AR antagonist propranolol and the β(2)-AR inverse agonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol hydrochloride (ICI-118,551). Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, peroxisome proliferator-activated receptor γ coactivator 1α, and multiple genes involved in the mitochondrial electron transport chain (F0 subunit 6 of transmembrane F-type ATP synthase, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 6, and NADH dehydrogenase [ubiquinone] 1β subcomplex subunit 8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified nisoxetine from the Sigma Library of Pharmacologically Active Compounds and two compounds from the ChemBridge DIVERSet that increased mitochondrial respiratory capacity. These data provide compelling evidence for the use and development of β(2)-AR ligands for therapeutic MB.

Figures

Fig. 1.
Fig. 1.
Formoterol exposure increases uncoupled OCR and mtDNA copy number in RPTC and AFC. A, RPTC were exposed to isoproterenol (nonspecific β-AR agonist), BRL 37344 (β3-AR agonist), and formoterol (β2-AR agonist) for 24 h and evaluated for changes in FCCP-OCR. B, AFC were exposed to isoproterenol (nonspecific β-AR agonist), BRL 37344 (β3-AR agonist), and formoterol (β2-AR agonist) for 24 h and evaluated for changes in FCCP-OCR. C, RPTC were exposed to the nonspecific β-AR agonists dopamine, epinephrine, and norepinephrine for 24 h and evaluated for changes in FCCP-OCR. D, RPTC were exposed to 30 nM formoterol for 24 h and evaluated for changes in mtDNA copy number relative to DMSO controls. Data are represented as mean ± S.E.M. of four biological replicates. *, P < 0.05.
Fig. 2.
Fig. 2.
Formoterol exposure induced the expression of mitochondrial genes and mtDNA copy number in the kidney cortex of CB57BL/6 mice. A, mRNA expression was evaluated in the kidney cortex of CB57BL/6 mice 24 h after a single intraperitoneal injection with 100 μg/kg formoterol. B, mRNA expression was evaluated in the kidney cortex of CB57BL/6 mice 72 h after daily repeated intraperitoneal injections with 100 μg/kg formoterol. C, mtDNA copy number was evaluated in the kidney cortex 24 and 72 h after daily repeated intraperitoneal injection with 100 μg/kg formoterol. Values indicate fold change relative to DMSO controls. Data are represented as mean ± S.E.M. of three to six biological replicates. *, P < 0.05.
Fig. 3.
Fig. 3.
Formoterol exposure induced the expression of mitochondrial genes and mtDNA copy number in the hearts of CB57BL/6 mice. A, mRNA expression was evaluated in the hearts of CB57BL/6 mice 24 h after a single intraperitoneal injection with 100 μg/kg formoterol. B, mRNA expression was evaluated in the hearts of CB57BL/6 mice 72 h after daily repeated intraperitoneal injections with 100 μg/kg formoterol. C, mtDNA copy number was evaluated in the heart 24 and 72 h after daily repeated intraperitoneal injection with 100 μg/kg formoterol. Values indicate fold change relative to DMSO controls. Data are represented as mean ± S.E.M. of three to six biological replicates. *, P < 0.05.
Fig. 4.
Fig. 4.
Cheminformatic analysis of formoterol identified nisoxetine, which induces MB in RPTC. A, RPTC were exposed to formoterol and nisoxetine (10–300 nM) for 24 h and examined for changes in FCCP-OCR. Values indicate a percentage of fold change relative to DMSO controls. Data are represented as mean ± S.E.M. of four biological replicates. B, pharmacophore based on the alignment of formoterol and nisoxetine. Formoterol and nisoxetine aligned with superimposed chemical features. F1 is a proton acceptor, F2 and F3 are hydrophobic, F4 is a mixed feature with a cationic and proton donor, and F5 and F6 are mixed features with aromatic or hydrophobic characteristics. F1 and F4 were marked as essential while requiring that at least five features matched the model. C, formoterol aligned with superimposed pharmacophore.
Fig. 5.
Fig. 5.
The effects of formoterol and nisoxetine are inhibited by β2 antagonism. A, RPTC were pre-exposed to the β-AR antagonist propranolol (5 nM) 1 h before exposure to 30 nM formoterol or nisoxetine and evaluated for changes in FCCP-OCR. B, RPTC were pre-exposed to the β2-AR inverse agonist ICI-118,551 (3 and 10 nM) 1 h before exposure to 30 nM formoterol or nisoxetine and evaluated for changes in FCCP-OCR. Values indicate a percentage of fold change relative to DMSO controls. Data are represented as mean ± S.E.M. of four biological replicates. *, P < 0.05.
Fig. 6.
Fig. 6.
Formoterol pharmacophore identified two ChemBridge compounds that induce MB in RPTC. A, RPTC were exposed to ChemBridge compounds 1, 2, and 3 (10–300 nM) for 24 h and evaluated for changes in FCCP-OCR. Values indicate a percentage of fold change relative to DMSO controls. Data are represented as mean ± S.E.M. of four biological replicates. B, refined pharmacophore based on the alignment of formoterol, nisoxetine, CB2, and CB3. In this alignment, F1 is a proton acceptor, F2 and F3 are hydrophobic, F4 is a mixed feature with a proton donor and a cationic or proton acceptor, and F5 and F6 are mixed features with aromatic or hydrophobic characteristics. F7 is a unique hydrophobic feature found in nisoxetine and CB2. F1, F2, F4, F5, and F6 are essential features. C, nisoxetine aligned with superimposed pharmacophore.

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