Fig 2: Effects of high levels of VEGF-B on skeletal muscle fiber types. (A) RT‒qPCR was used to detect the expression of MyHC-1, MyHC-2a, MyHC-2b, and MyHC-2x on the 6th day of differentiation in C2C12 myoblast cells treated with 100 ng/mL VEGF-B167 and VEGF-B186. n = 3. *P < 0.05, **P < 0.01. (B) Representative images of My32 and NOQ staining on the 6th day of differentiation in C2C12 myoblast cells treated with 100 ng/mL VEGF-B167 or VEGF-B186. DAPI represents the nucleus. Bar = 100 µM. (C) Western blot of NOQ and My32 on the 6th day of differentiation in C2C12 myoblast cells treated with 100 ng/mL VEGF-B167 and VEGF-B186. (D-E) Quantitative assays for the expression of NOQ and My32 in (C). n = 3. *P < 0.05, **P < 0.01

Fig 3: Role of VEGF-B in myoblast differentiation, fusion and myotube formation. (A) RNA-seq analysis of differentiation- and fusion -associated genes in GA tissue from WT and Vegfb−/− mice fed a ND or HFD. n = 3. (B) Representative images of MyHC staining of C2C12 myoblast cells differentiated under different doses of VEGF-B167 and VEGF-B186 (1 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, or 100 ng/mL) with continuous single-dose administration for 6 days. Green fluorescence represents MyHC, and DAPI represents the nucleus. Bar = 100 µM. (C-D and F) Differentiation indices (C), fusion indices (D), and myotube numbers (F) on day 6 of C2C12 myoblast cell differentiation under different doses of VEGF-B167 and VEGF-B186 with continuous single-dose administration. (F) Quantitative assay for the number of MyHC-positive myotubes containing more than 2 nuclei in differentiated C2C12 myoblast cells treated with different doses of VEGF-B167 and VEGF-B186 on day 6. n = 5. *P < 0.05, **P < 0.01 compared with the Ctrl group; #P < 0.05, ##P < 0.01 compared with the 1 ng/mL group; $P < 0.05, $$P < 0.01 compared with the 10 ng/mL group; &P < 0.05, &&P < 0.01 compared with the 20 ng/mL group; @P < 0.05 compared with the 50 ng/mL group in (C-D and F). (E) Representative images of MyHC staining in differentiated C2C12 myoblast cells treated with 100 ng/mL VEGF-B167 or VEGF-B186 for 2, 4 or 6 days. Bar = 100 µM. The number at the bottom right of the image indicates the number of myotubes

Fig 4: Schematic diagram of this work. (A) VEGF-B acts as a bridge between skeletal muscle type and high-fat diet-induced hyperglycemia in vivo. (B) Matched with high-fat diet-induced hyperglycemia accompanied by increased content of slow-twitch type I fibers within the skeletal muscle of VEGF-B deficient high-fat diet-fed mice in vivo, VEGF-B186, which was continuously administered at a single high dosage in vitro, inhibited myoblast differentiation/fusion and myofiber formation and glucose utilization via the PKA-NFATs signaling pathway

Fig 5: VEGF-B186 inhibits myoblast differentiation/fusion and slow-twitch myofiber formation through the PKA-NFAT-MyoG/MEF2C signaling pathway. (A) Heatmap showing the expression of NFAT signaling pathway genes in the gastrocnemius muscle tissue of WT and Vegfb−/− mice fed a ND or HFD. (B) Quantitative analysis of NFATc1 and NFATc2 expression in (A). n = 3. **P < 0.01. (C-E) Western blot detection of Nfatc1 and Nfatc2 proteins in the tibialis anterior muscle of WT-ND, Vegfb−/−-ND, WT-HFD and Vegfb−/−-HFD mice. n = 3. *P < 0.05, **P < 0.01. (F-G) Western blot detection of P-Nfatc1/c2 in the cytoplasm and NFATc1/c2 in the cell nucleus on the 6th day of differentiation in C2C12 myoblast cells treated with 100 ng/mL VEGF-B186 with or without N6-Bz-cAMP (10− 5 mol/L) and 8-Bromo-cAMP (10− 5 mol/L). n = 3. *P < 0.05, **P < 0.01. (H) Western blot detection of MyHC and NOQ on the 6th day of differentiation in C2C12 cells treated with 100 ng/mL VEGF-B186 with or without AdNFATc1/c2. n = 3. *P < 0.05, **P < 0.01. (I) Representative images of MyoG + and MEF2C + staining on the 6th day of differentiation in C2C12 myoblast cells treated with 100 ng/mL VEGF-B186 with or without AdNFATc1/c2. DAPI represents the nucleus. Bar = 100 µM