Fig 2: AAV1-FGF21 treatment increases energy expenditure(A) Food intake of male mice fed an HFD and treated with AAV1-FGF21 (n = 2–3 cages/group). (B) Energy expenditure was measured with an indirect open circuit calorimeter at different time points, during light and dark cycles (n = 6–10/group). (C) Interscapsular BAT (iBAT) weight of AAV-FGF21-treated male mice at different ages (n = 5–10/group). (D) Representative images of hematoxylin and eosin staining of iBAT sections. Scale bars, 100 μm. (E–G) Quantification of the expression of the non-shivering thermogenesis markers Ucp1 (E), Cidea (F), and Elovl3 (G) in iBAT at age 70 weeks (n = 5–10/group). (H) Representative tracks during the open field test of male mice at age 8 and 70 weeks. (I–P) Assessment of locomotor activity (I–N) and anxiety parameters (O and P) through the open-field test (n = 5–15/group). Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way analysis of variance (ANOVA) with Tukey’s (A, E–G, I–P) or Bonferroni (B and C) multiple comparison test. FC, fold change; w, weeks.

Fig 3: AAV1-FGF21 biodistribution and biological activity in dogsHealthy Beagle dogs (dog-1 and dog-2) were treated with 7 × 1012 vg/kg of AAV1-canine optimized FGF21 and followed up for 4 months. (A) Vector genome copy number (left panel, blue) and canine optimize FGF21 (coFGF21) expression (right panel, orange) were analyzed in tissue punches from multiple regions of the skeletal muscle and the liver obtained during necropsy of dogs. (B) Schematic representation indicating the mean coFGF21 expression in hindlimb skeletal muscles. Image courtesy of IMAIOS️ (Micheau A, Hoa D, e-Anatomy, www.imaios.com, https://doi.org/10.37019/e-anatomy). (C) Levels of biologically active FGF21 in fasted conditions measured using a cell-based reporter gene assay (iLite). (D) Hepatic expression levels of KLB (n = 2–3/group). (E) Serum triglyceride levels pre-AAV and 4 months post-AAV (n = 1–3). (F and G) Hepatic expression levels of ACADM (F) and ACADL (G) (n = 2–3/group). (H) Serum adiponectin levels pre-AAV and 4 months post-AAV (n = 1–3). (I–L) Quantification of ADIPOQ (I), UCP1 (J), ELOVL3 (K), and PPARGC1A (L) expression in perirenal (prWAT) and gluteal WAT (gWAT) (n = 2–3/group). (M) Representative images of the hematoxylin and eosin staining of prWAT sections. Insets show multilocular adipocytes in prWAT of AAV-FGF21-treated dogs. Scale bars, 100 μm. Inset scale bars, 25 μm. Data are presented as mean ± SD. Data were analyzed using a Mann-Whitney two-tailed test. coFGF21, canine optimized FGF21; ND, non-detected; vg/dg, vector genomes/diploid genome; AU, arbitrary units; FC, fold change; G, gluteus; T, tensor; Q, quadriceps; V, vastus; LLL, left lateral lobe; LML, left medial lobe; RML, right medial lobe.

Fig 4: AAV-mediated skeletal muscle gene transfer of FGF21 reverses hepatic steatosisEight-week-old male mice were fed an HFD. Twenty weeks later, mice were treated intramuscularly with AAV-FGF21 vectors and remained in HFD feeding for all experimental periods. Non-injected chow and HFD-fed mice were used as controls. (A) Body weight follow-up (n = 9–10/group). (B) Body weight gain in HFD-fed groups from weeks 28–70 of age (n = 5–10/group). (C) Serum FGF21 levels at different time points (n = 5–11/group). (D) Quantitative PCR analysis of murine optimized Fgf21 (moFgf21) expression in the three injected skeletal muscles and in the liver in 70-week-old male mice (n = 5–7/group). (E) Hepatic expression of Klb (n = 5–10/group). (F) Representative macroscopic images of the liver from control HFD-fed (left) and AAV-FGF21-treated (right) male mice at age 50 weeks. (G) Representative images of hematoxylin and eosin staining of liver sections at age 50 and 70 weeks. Scale bars, 100 μm. (H) Liver weight at different time points (n = 5–11/group). (I and J) Liver triglyceride (I) and cholesterol (J) content (n = 5–11/group). (K and L) Serum ALT (K) and aspartate aminotransferase (AST) levels (L) (n = 5–9/group). Data are presented as mean ± SD; ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. AU, arbitrary units; ND, non-detected; FC, fold change; w, weeks; ꝉ, death due to natural causes.

Fig 5: Reversal of WAT hypertrophy and inflammation by AAV1-FGF21 treatment(A) Immunohistochemical analysis of MAC2 in eWAT sections. Red arrows indicate crown-like structures. Scale bars, 100 μm. (B) Morphometric analysis of the mean adipocyte area in eWAT (n = 4/group). (C and D) Weight of eWAT (C) and iWAT (D) depots (n = 5–10/group). (E and F) Serum leptin (E) and adiponectin (F) levels (n = 5–10/group). (G–I) Expression levels of the inflammatory markers Cd68 (G), F4/80 (H), and Tnfa (I) in eWAT (n = 5–10/group). Data are presented as mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. FC, fold change; w, weeks.