Phloretin Suppresses Preadipocyte Differentiation by Targeting PPARγ/Transferrin to Activate Ferroptosis Signaling Pathway

ABSTRACT Phloretin, a plant‐derived dihydrochalcone bioactive compound, potentially modulates preadipocyte differentiation, although this remains controversial and requires further clarification. To clarify this point, herein, phloretin gavage inhibited mice obesity, reduced adipocyte size, and ameliorated serum lipid parameters, including triglycerides. In vitro, phloretin suppressed the differentiation of 3T3‐L1 and bovine preadipocytes, decreasing lipid droplet content and reducing CEBPα and PPARγ levels. Moreover, during differentiation, glutathione peroxidase (GSH‐Px) ultimately decreased with elevations in malondialdehyde (MDA) and Fe 2+ levels, along with acyl‐CoA synthetase long‐chain family member 4 ( ACSL4 ), P53 , and SLC7A11 expression, indicating differentiation occurring under mild oxidation and slight ferroptosis activation. Similar to (1 S ,3 R )‐RSL3, phloretin further stimulated the ferroptosis pathway by inhibiting glutathione reductase (GR) and GSH‐Px, increasing the content of MDA and Fe 2+ . Simultaneously, the suppression of Fth1 , Slc7a11 , and Sod2 transcription, significantly elevated HMGB1 protein, and markedly downregulated glutathione peroxidase 4 (GPX4) and TFR protein collectively confirmed that phloretin activated the ferroptosis signaling pathway to inhibit differentiation. Further phosphoproteomic analysis identified dm‐phosphorylation sites (p‐eEF2K[S365], p‐AMPKα1[S491], and p‐CaMKK2[S495]) enriched in AMPK/MAPK signaling pathways and transferrin receptor binding. Further molecular docking simulations revealed that phloretin binds to PPARγ or transferrin to activate ferroptosis pathway, revealing the potential crosstalk between ferroptosis signaling pathway and adipogenic differentiation processes.