Fig 2: FGF21 circulating levels in human patients(A and B) Serum FGF21 levels were measured in a cohort of 46 lean and 12 overweight individuals and in a cohort of approximately 500 highly obese and insulin-resistant patients, grouped by body mass index (BMI) (A) and in a cohort of 20 biopsy-proven MASH patients and grouped by fibrosis stage (B). Gray bar indicates FGF21 circulating levels corresponding to 90% of healthy individuals (0–0.26 ng/mL). The 2-fold limit of the normal range displayed by lean individuals (0.53 ng/mL) is indicated with the dashed line. n, number of individuals.

Fig 3: AAV1-FGF21 improves insulin sensitivity(A) Fed and fasted serum insulin in male mice at age 70 and 38 weeks, respectively (n = 5–9/group). (B) Fed and fasted blood glucose in male mice at age 48 and 38 weeks, respectively (n = 8–10/group). (C) Insulin sensitivity was determined after an intraperitoneal insulin injection (0.75 units/kg) at age 59 weeks in male mice. Results were calculated as percentage of initial blood glucose levels (n = 9–10/group). (D) Representative images of insulin immunostaining in pancreas sections from AAV-FGF21-treated male mice. Scale bars, 500 μm. (E) Quantification of β cell mass in male mice at age 70 weeks (n = 5/group). (F) Fasted glucagon levels in male mice, 12 weeks post-AAV administration (n = 8–10/group). (G) Forelimb grip strength in female mice (n = 6–9/group). (H) Time before falling during the rotarod test performed in female mice (n = 6–9/group). (I and J) Discrimination index (I) and exploration time (J) during novel object recognition (NOR) test performed in female mice (n = 6/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 multiple comparison test (A, B and E–I) or Student’s two-tailed t test (J). In (C), #p < 0.05, ##p < 0.01, ###p < 0.001 versus chow-fed control group and $$p < 0.01, $$$p < 0.001 versus HFD-fed control group by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. BW, body weight; N, Newtons.

Fig 4: Muscle-derived FGF21 counteracts hepatic fibrosis and halts development of liver tumors(A) Representative images of Picrosirius red (PSR) staining of liver sections showing liver fibrosis in red. Scale bars, 50 μm. (B) Histomorphometric quantitative assessment of liver fibrosis (PSR) (n = 2–4/group). (C and D) Expression levels of the liver fibrosis markers Col1a1 (C) and Col3a1 (D) (n = 5–10/group). (E–G) Expression levels of the genes involved in extracellular matrix deposition Mmp12 (E), Mmp13 (F), and Timp1 (G) (n = 5–10/group). (H–K) Expression levels of the marker of activated hepatic stellate cells (HSCs) a-Sma (H) and key cytokines involved in HSCs activation Tfgb (I), Pdgfa (J), and Pdgfb (K) (n = 5–10/group). (L–P) Expression levels of the hepato-cellular carcinoma markers Afp (L), Ly6d (M), Krt19 (N), Golm1 (O), and Cd44 (P), in non-tumoral liver parenchyma (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.

Fig 5: Treatment with AAV-FGF21 vectors reverts hepatic inflammation(A) Representative images of liver sections immunostained against the macrophage marker MAC2. Red arrows indicate MAC2+ cells. Scale bars, 100 μm. (B–G) Expression levels of the hepatic inflammatory markers Cd68 (B), F4/80 (C), Ccl2 (D), Ccl3 (E), Ccl5 (F), and Tnfa (G) (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.