Fig 2: CpG site-specific DNA methylation analyses at the adiponectin gene locus in adipose tissues and blood cells from mothers with NGT vs. GDM and their offspring. Schematic illustration of the adiponectin (ADIPOQ) gene locus, including characterized transcription factor binding sites (e.g., SRE, PPRE, C/EBP), and analyzed DNA methylation assays (R1-R3) (a). Percent DNA methylation is shown for each individual CpG site (numbering follows 5′ to 3′), analyzed per assay (R1–R3) for subcutaneous adipose tissue (SAT; b), visceral adipose tissue (VAT; c), maternal blood (MB; d), and cord blood (CB; e) in the normal glucose tolerant (NGT; open boxes; n = 30) vs. gestational diabetes mellitus group (GDM; red boxes; n = 22–25). Group comparisons in cord blood samples were adjusted for newborn sex. Box-whisker plots show the minimum and maximum values. Pearson’s correlation coefficients (R) were calculated to determine the relationships between DNA methylation of significant CpG sites and respective ADIPOQ mRNA levels in VAT across the whole cohort (f, g). Gene expression of ADIPOQ was normalized to peptidylprolyl isomerase A (PPIA). NGT, open circles; GDM, red circles. AU, arbitrary units. *P < 0.05, **P < 0.01, ***P < 0.001

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.