Temporal regulation of activin, follistatin, and growth differentiation factor 15 in the liver and circulation during high-fat diet-induced fatty liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) develops over time, yet most hepatokine studies are cross-sectional. We investigated the temporal regulation of the activin-follistatin axis and growth differentiation factor 15 (GDF15) in high-fat diet (HFD)-induced fatty liver disease. Mice were fed an HFD or normal chow for 8, 20, or 30 weeks. Plasma, hepatic mRNA, and hepatic protein were measured by ELISA, real-time PCR, and immunoblotting. HFD increased body and liver weights and hepatic triglyceride content throughout. A central finding was discordance between compartments: hepatic Inhba mRNA was elevated at 8 and 30 weeks, yet plasma and hepatic activin A protein both peaked at 20 weeks, indicating post-transcriptional regulation; hepatic follistatin protein decreased at 20 and 30 weeks despite an early rise in Fst mRNA, while circulating follistatin and follistatin-like 3 (FSTL3) were unchanged. The activin receptors ACVR2B and ACVR1 showed distinct protein kinetics, with sustained ACVR2B elevation and a transient ACVR1 peak at 20 weeks, potentially favoring canonical activin signaling. Together these changes shifted the balance toward activin, increasing plasma activin A-to-follistatin and activin A-to-FSTL3 ratios at 20 weeks and the phospho/total-SMAD2/3 ratio at 20 and 30 weeks. Hepatic Gdf15 mRNA was induced throughout, whereas plasma GDF15 (measured at 8 and 20 weeks) was increased at both and correlated positively with plasma activin A. These findings reveal time-dependent, level-discordant regulation of the activin-follistatin axis and GDF15 in HFD-induced fatty liver, providing a temporal reference unavailable from cross-sectional studies.