Activation of 5‐hydroxytryptamine 1F Receptor Induces Mitochondrial Biogenesis Through Dual Mechanisms
Authors: Gibbs Whitney Sharee , Beeson Craig C. , Schnellmann Rick G 
Published: November 2023

BackgroundMitochondrial dysfunction limits repair mechanisms and restoration pathways required for the recovery of cellular functions following acute organ damage. Our laboratory has demonstrated that the 5‐hydroxytrptamine 1F (5‐HT1F) receptor agonist LY344864 (LY) induces new, functional mitochondria through mitochondrial biogenesis (MB), resulting in accelerated renal recovery following ischemia/reperfusion induced acute kidney injury in mice. The goal of this study was to determine the signaling pathways involved in LY induced MB.MethodsMaximal mitochondrial respiration (FCCP‐OCR, a marker of MB) was measured in renal proximal tubule cells (RPTC) using a Seahorse instrument. Signaling pathways were explored using pharmacological inhibitors and immunoblot analysis. cGMP was measured using an ELISA.ResultsLY increased p‐Akt at 15–30 min and this increase was attenuated by the Gβγ protein complex inhibitor gallein. eNOS, a downstream target of Akt, was measured following LY in the presence and absence of gallein or the Akt inhibitor GDC‐0068 (GDC). LY increased p‐eNOS at 1 hr that was attenuated by gallein and GDC. cGMP accumulation was also increased at 1 hr. Vasodilator‐stimulated phosphoprotein (VASP) phosphorylation was measured as a marker of protein kinase G (PKG) activity in the presence and absence of the guanylate cyclase inhibitor ODQ or the protein kinase G inhibitor KT5823. LY increased p‐VASP at 1–2 hr and this increase was attenuated by ODQ and KT5823. In addition, phosphorylation of serine/threonine sites on peroxisome proliferator‐activated receptor gamma coactivator‐1α (PGC‐1α), the master regulation of MB, increased at 2 hr. Pretreatment with gallien, GDC, the nitric oxide synthase inhibitor L‐NAME, ODQ, or KT5823 attenuated LY‐induced increases in FCCP‐OCR.In contrast to the Akt/eNOS/cGMP/PKG/PGC‐1α pathway, p‐ERK1/2 was decreased at 1 hr following LY exposure and gallein and GDC blocked the reduction of p‐ERK1/2, indicating that both Gβγ and Akt mediate LY reduction of p‐ERK1/2. c‐Raf phosphorylation (Ser 259) by Akt is known to reduce MEK/ERK signaling. A 30 min exposure to LY elevated p‐c‐raf (Ser 259), which was prevented by GDC, indicating Akt‐dependent c‐raf phosphorylation. Phosphorylation of FOXO3a at serine 294, a downstream target of ERK1/2 and transcriptional regulator of PGC‐1α, prevents nuclear translocation of FOXO3a and reduces the transcription of genes such as PGC‐1α. Following 2 hr LY exposure, FOXO3a phosphorylation was reduced, suggesting increased FOXO3a nuclear translocation and transcription of PGC‐1α.ConclusionThis study reports the novel finding that the 5‐HT1F receptor induces renal MB through Gβγ heterodimer‐dependent activation of Akt/eNOS/cGMP/PKG/PGC‐1α and repression of Akt/c‐raf/ERK/FOXO3a pathways. We also identified Akt as the link between the stimulatory and inhibitory pathways, and that the stimulatory is required for MB. These findings support the use of 5HT1F receptor agonists for treatment of mitochondrial dysfunction in organ injuries.Support or Funding InformationNIH: R01GM084147 (R.G.S.), 5T32‐DK083262 (W.S.G.); VA: BX‐000851 (R.G.S.)