IF AAV9-mediated hepatic FGF21 overexpression (single tail-vein injection, ~1×10¹¹ vg/mouse, producing sustained supraphysiological circulating FGF21) is combined with intermittent oral fisetin (100 mg/kg, 5 consecutive days every 4 weeks — a validated senolytic dosing schedule) and administered to 20-month-old male C57BL/6J mice for 16 weeks,

THEN the combination arm will show, relative to either monotherapy or vehicle control: (1) ≥40% greater restoration of cold-stimulated (4°C, 7-day) de novo beige adipocyte formation in inguinal white adipose tissue (iWAT), quantified by UCP1+ cell density and oxygen consumption rate (OCR) in freshly isolated SVF adipocytes; (2) ≥50% reduction in p16^INK4a^/p21^CIP1^ double-positive cells within the iWAT stromal vascular fraction (SVF); (3) ≥30% improvement in mitochondrial membrane potential (JC-1 ratio) in SVF progenitors; and (4) ≥25% reduction in plasma circulating cell-free mitochondrial DNA (cf-mtDNA) as an integrated SASP-damage biomarker — with the combination outperforming monotherapy in all four endpoints simultaneously,

BECAUSE the following causal chain connects the intervention to the outcomes:

1. FGF21 activates the SIRT1 → mTOR-inhibition → TFEB nuclear translocation axis in adipose-resident progenitor cells, enhancing autophagy flux and selectively degrading fragmented, depolarized mitochondria in pre-senescent beige progenitors that retain some autophagic capacity. (FGF21 activates SIRT1, inhibits mTOR phosphorylation, and promotes TFEB nuclear translocation to enhance lysosomal biogenesis and autophagy flux, and autophagy flux inhibition with chloroquine reverses these cytoprotective effects)[https://doi.org/10.1038/s41419-021-04157-x]

2. Chloroquine-reversibility of FGF21's benefit establishes mitophagy flux—not transcriptional reprogramming—as the obligate effector, meaning FGF21's repair action mechanistically requires a functional lysosomal pathway. (autophagy flux inhibition with chloroquine reversed FGF21's protective effects)[https://doi.org/10.1038/s41419-021-04157-x] This localizes the MitoSENS repair action to the lysosome-autophagosome axis.

3. Fully senescent p16^INK4a+^ beige progenitors in aged iWAT are refractory to FGF21-driven mitophagy induction because deep senescence is characterized by mTOR hyperactivation and suppressed autophagy flux — the same pathway FGF21 requires. These cells represent a "damage floor" that FGF21 cannot clear. (Activating the senescence pathway, genetically or pharmacologically, in young beige progenitors induces premature senescence and blocks their potential to form cold-induced beige adipocytes)[https://doi.org/10.1016/j.cmet.2016.10.023]

4. Fisetin selectively eliminates p16^INK4a+^ senescent cells from the beige progenitor niche, decompressing the niche and removing the primary block to progenitor activation that FGF21 cannot overcome. (Fisetin reduced senescence markers across multiple tissues in progeroid and old mic...

SENS category: RepleniSENS

Key references: • doi.org/10.1038/s41419-021-04157-x] • doi.org/10.1016/j.cmet.2016.10.023] • doi.org/10.1016/j.ebiom.2018.09.015] • doi.org/10.1038/s41467-020-18039-x] • doi.org/10.1073/pnas.1910073116]