Fig 1: Fasting induced adipose-specific VEGF expression and liver Fgf21 expression. Twelve-week-old male C57BL/6J mice were either fed with chow diets ad libitum or fasting for various time periods (6 h, 12 h, and 24 h) as indicated. (A) Serum VEGF levels and (B) the mRNA expression of Vegfa in epididymal WAT (eWAT) (B), subcutaneous iWAT (C), interscapular BAT (D), liver (E), and muscle (F) as determined by real-time PCR analysis. The protein levels of VEGF in eWAT (G) and iWAT (H) as determined by western blot; (I) serum FGF21 levels as determined by enzyme-linked immunosorbent assay; and real-time transcription PCR analysis for Fgf21 mRNA expression levels of liver (J), eWAT (K), iWAT (L), BAT (M), and muscle (N). Serum FFA (O) and ketone bodies (P) levels. Data are mean ± SEM; n = 6/group. Statistical significance was evaluated by 1-way ANOVA with Tukey’s test for multiple comparisons to determine differences between each group. Labeled means without a common letter differ, P < 0.05. Abbreviations: BAT, brown adipose tissue; eWAT, epididymal white adipose tissue; FFA, free fatty acid; FGF21, fibroblast growth factor 21; iWAT, inguinal white adipose tissue; VEGF, vascular endothelial growth factor.
Fig 2: Intermittent fasting induced adipose-VEGF expression and angiogenesis depend on liver FGF21 signaling. Mice were fed with HFD ad libitum or time-restricted access to food for 16 weeks. HA means WT mice eating a high-fat diet with ad libitum, HT means WT mice eating a high-fat diet with time-restricted access to food, KOHA means FGF21 LKO mice eating a high-fat diet with ad libitum, KOHT means FGF21 LKO mice eating a HFD with time-restricted access to food. (A) Schematic illustration of the experimental design. (B) Body weight. (C) Representative HT mice were remarkably leaner than the HA mice. (D) Body composition was evaluated by EchoMRI. (E) Serum FGF21 levels as determined by enzyme-linked immunosorbent assay. (F) A representative macroscopic image illustrating increased vascularization in iWAT of HT mice, compared to HA mice but not in FGF21 LKO mice. (G) Real-time quantitative PCR analysis for mRNA expression levels of Vegfa in eWAT and iWAT. Representative protein levels for VEGF in eWAT (G) and iWAT (I). (J) Immunofluorescence staining of CD31 (scale bar, 100 mm) in eWAT and iWAT, illustrating IF increased vascularization in WT mice but not in FGF21 LKO mice. Data are mean ± SEM; n = 6–8/group. Statistical significance was evaluated by the 2-way ANOVA test and the Tukey’s test for multiple comparisons to determine differences between each group. HA vs HT, *P < 0.05, **P < 0.01; labeled means without a common letter differ, P < 0.05.
Fig 3: FGF21 promoted expression and accumulation of VEGF in WAT. The relative mRNA abundance of Vegfa in tissue, adipocytes and stromal vascular fraction isolated from eWAT (A) and iWAT (B) at fed or 24 h of fasting. Twelve-week-old male WT (FGF21fl/fl) and FGF21 LKO mice were fasted for 24 h; the Vegfa mRNA expression in eWAT and iWAT (C). Quantitative reverse transcription PCR analysis for Vegfa mRNA expression in eWAT (D), iWAT (E), and BAT (F) at the indicated time points after tail vein injection of rmFGF21 (1 mg/kg). The protein levels of VEGF at various time points after mice receiving a delivery of rmFGF21 with tail vein injection in eWAT (G) and iWAT (H). n = 6/group. Statistical significance was evaluated by unpaired Student’s t test. *P < 0.05, **P < 0.01, versus control; labeled means without a common letter differ, P < 0.05. Abbreviation: SCV, stromal vascular fraction.
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