Structural and pharmacological basis for the induction of mitochondrial biogenesis by formoterol but not clenbuterol

Abstract

Mitochondrial dysfunction is associated with numerous acute and chronic degenerative diseases. The beta-2 adrenergic receptor (β2AR) agonist formoterol induces mitochondrial biogenesis (MB), but other β2AR agonists, such as clenbuterol, do not. We sought to identify the MB signaling pathway of formoterol and the differences in signaling between these two ligands that result in the differential induction of MB. While formoterol and clenbuterol increased cAMP, only formoterol increased the phosphorylation of Akt and its downstream target eNOS. The increase in Akt phosphorylation was Gβγ- and PI3K-dependent, and the increase in eNOS phosphorylation was Gβγ- and Akt-dependent. Only formoterol increased cGMP. Formoterol induced MB as measured by increases in uncoupled cellular respiration and PGC-1α and NDUFS1 mRNA expression and was blocked by inhibitors of Gβγ, Akt, NOS, and soluble guanylate cyclase. To identify distinct receptor-ligand interactions leading to these differences in signaling, we docked formoterol and clenbuterol to six structures of the β2AR. Compared to clenbuterol, the methoxyphenyl group of formoterol interacted more frequently with V114 and F193, while its formamide group interacted more frequently with C191. These data indicate that the unique structural features of formoterol allow it to interact with the β2AR to activate the Gβγ-Akt-eNOS-sGC pathway to induce MB.

Pharmacological Stimulation of Mitochondrial Biogenesis Using the Food and Drug Administration-Approved β2-Adrenoreceptor Agonist Formoterol for the Treatment of Spinal Cord Injury

“…Based on increases in mtDNA and mitochondrial gene and protein expression, our studies have shown that formoterol induces MB in the kidney, heart, skeletal muscle, and spinal cord of mice, while clenbuterol has not been found to induce MB. 26,27,32,55 Recent work from our laboratory has attributed this difference to unique structural features of the two agonists resulting in distinct receptor-ligand interactions and divergent signaling pathways. Specifically, formoterol is able to extend across the ADRB2 binding pocket, leading to activation of the Gbc-Akt-eNOS-sGC pathway, which is necessary for the induction of MB; clenbuterol and other non-mitochondrially biogenic ADRB2 agonists do not activate this pathway.…”

“…Specifically, formoterol is able to extend across the ADRB2 binding pocket, leading to activation of the Gbc-Akt-eNOS-sGC pathway, which is necessary for the induction of MB; clenbuterol and other non-mitochondrially biogenic ADRB2 agonists do not activate this pathway. 32 Therefore, despite both formoterol and clenbuterol being ADRB2 agonists, dissimilar structures and signaling leading to MB with formoterol has a greater therapeutic potential for the management of SCI.…”

“…Specifically, formoterol contains a longer methoxyphenyl group, which clenbuterol lacks, that extends across the ADRB2 binding pocket, and leads to activation of the Gbc-Akt-eNOS-sGC pathway and the subsequent induction of MB. 32 Therefore, formoterol is not only a more potent, selective, and long-acting ADRB2 agonist than clenbuterol, but also induces MB, 26,33 giving this pharmacological agent the potential to alleviate multiple aspects of injury progression and promote recovery post-SCI. Given the importance of ischemia during secondary injury and the detrimental mitochondrial dysfunction that accompanies SCI, this study sought to determine the therapeutic potential of formoterol-mediated MB on functional recovery using an impactor-induced contusion mouse model of SCI.…”

“…7B). Analysis of all data revealed a significant effect of both formoterol treatment (F [2,32] = 98.05, p < 0.0001) and days post-injury (F [15,480] = 37.76, p < 0.0001), as well as a significant interaction (F[30,480] = 16.52, p < 0.0001). While all injured mice lost a comparable percentage of body weight post-injury, formoterol-treated mice gained body weight compared with vehicle-treated mice beginning on day 7, and reached a weight no different than their initial starting weight by day 11.…”

Proximal Tubule β2-Adrenergic Receptor Mediates Formoterol-Induced Recovery of Mitochondrial and Renal Function after Ischemia-Reperfusion Injury

“…We reported that formoterol treatment restored renal function with concomitant increases in mitochondrial protein expression and function after AKI in mice (Jesinkey et al, 2014). We also elucidated the mechanism of formoterol-induced MB in RPTCs (Cameron et al, 2017). ABBREVIATIONS: AKI, acute kidney injury; b 2 AR, b-2 adrenergic receptor; DMSO, dimethylsulfoxide; Drp1, dynamin-related protein 1; IRI, ischemia-reperfusion injury; KIM-1, kidney injury marker-1; KO, knockout; MB, mitochondrial biogenesis; Mfn2, mitofusin 2; NDUFS1, NADH: ubiquinone oxidoreductase core subunit S1; PCR, polymerase chain reaction; PGC-1a, peroxisome proliferator-activated receptor gamma coactivator-1a; RPTC, renal proximal tubule cell; SCr, serum creatinine; WT, wild-type.…”