Fig 1: Systemic AAV delivery of combination gene therapy reverses symptoms of obesity for mice on an HFD. (A) Venn diagram of the combinations of gene therapies explored as well as the color coding for all subsequent graphs. FK, FGF21 + aKlotho. (B) Weight of mice over time injected with a control vector AAV:GFP on an ND or an HFD, and mice on an HFD injected with individual or all combinations of AAV:FGF21, AAV:sTGFbR2, or AAV:aKlotho. n = 10 for AAV:GFP ND mice, and n = 12 for AAV:GFP and all therapy-treated HFD mice. (C) Phenotype exhibited by mice on an HFD with control vector (Right) and AAV:FGF21 vector (Left). (D) Long-term effect on mice receiving an HFD of the AAV:FGF21 therapy up to 250 d postinjection (n = 4). (E) Percentage weight change of 18-mo-old mice on an ND with starting weight ~40 g (n = 25 for each group). (F) CT scan of mice observing bone density after administration of AAV:FGF21 (n = 2 for each group). (G) Quantified muscle mass of MRI of whole mouse (n = 2 for each group). Statistical tests in B and E are 2-way ANOVA. Statistical tests in F and G are 2-sided t tests. Error bars represent SEM. C, control; F, FGF21; K, aKlotho; T, sTGFßR2; TF, sTGFßR2 + FGF21; TFK, sTGFßR2 + FGF21 + aKlotho; TK, sTGFßR2 + aKlotho. *P < 0.01 compared with AAV:GFP-HFD; **P < 0.0001 values compared with AAV:GFP-HFD.
Fig 2: Systemic AAV delivery of combination gene therapy stops progression of heart failure in an AAC mouse model. (A) FS quantification of ECHOs at baseline and 7, 30, and 90 d postsurgery. n = 10 for control (C), sTGFßR2 (T), sTGFßR2 + FGF21 (TF), and sTGFßR2 + FGF21 + aKlotho (TFK). n = 13 for sTGFßR2 + aKlotho (TK). (B) Representative ECHOs of mice at 3 mo post-AAC surgery. (C) Quantification of EF from ECHO. (D) Heart weight (HW) relative to body weight (BW) measured on day 90 post mortem. n for C = 6, n for T = 7, n for TF = 9, n for TK = 9, and n for TFK = 8. (E) Representative image of MTS hearts taken at 10× and stitched together. (F) Quantification of MTS-stained hearts. n for C = 10, n for T = 10, n for TF = 10, n for TK = 13, and n for TFK = 10. All images were taken at 10×, stitched together using Zen Zeiss software, and analyzed using custom MATLAB software that used color thresholding to separate different color pixels. Full ECHO data, including wall thickness, can be found in SI Appendix. Statistical tests in A and B are 2-way ANOVA, with P values representing comparison with AAV:GFP control mice over time. Statistical tests in D and F are 1-way ANOVA. Error bars represent SEM. F, FGF21. *P < 0.05; **P < 0.005; †P < 0.0002.
Fig 3: Systemic AAV delivery of combination gene therapy mitigates renal damage due to UUO. (A) Representative MTS kidneys for mice that underwent UUO surgery at day 7 for control, AAV:aKlotho, AAV:FGF21, and AAV:FGF21 + AAV:sTGFbR2 therapy groups. (B) Quantification of renal medullary atrophy of different therapy group kidneys. Photoshop was used to calculate the area of atrophy by tracing inner and outer edges and measuring pixel area. If there was a discontinuity in the shape edge, an ellipse was used for approximation. n values are as follows: control (C) = 7, sTGFßR2 (T) = 8, aKlotho (K) = 6, FGF21 (F) = 8, TK = 8, FK = 6, TF = 7, TFK = 6. (C) Representative images of SMA- and WGA-stained kidneys. (D) Quantification of the ratio of SMA- to WGA-stained kidney sections. n values: C = 5, T = 7, K = 7, F = 8, TK = 5, FK = 7, TF = 9, TFK = 7. All images were taken at 10×, stitched together using Zen Zeiss software, and analyzed using custom MATLAB software that used color thresholding to separate different color pixels. Statistical tests in B and D are 1-way ANOVA. P values compare each therapy group with AAV:GFP. Error bars represent SEM. *P < 0.05; **P < 0.01; †P < 0.001. FK, FGF21 + aKlotho.
Fig 4: Opposite effect of adipocyte-specific SERCA2 ablation on white and brown adipose tissue glucose uptake and mitochondrial function. (A) Total glucose uptake in chow-fed WT and adipocyte-specific SERCA2 KO mice. (B) Mitochondrial calcium measured with Rhod-2 dye and (C) ROS production measured with CM-H2DCF-DA dye in 3T3-L1 treated with vehicle or CPA with or without the mitochondrial calcium uptake inhibitor RU for 1 h. (D) Oxygen consumption rate (OCR) in 3T3-L1 treated with CPA or vehicle for 4 h. (E) Mitochondrial mass measured with Mitotracker Green dye. (F) Total OXPHOS (Complex I–V) and UCP1 protein levels (expressed as % of WT) in isolated mitochondria IWAT in chow-fed WT and adipocyte-specific SERCA2 KO mice. (G) OCR of the different mitochondrial complexes and (H) respiratory states analysed in isolated IWAT mitochondria from chow-fed WT and adipocyte-specific SERCA2 KO mice. (I) OCR parameters calculated in adipocytes differentiated from IWAT SVF from chow-fed WT and adipocyte-specific SERCA2 KO mice. (J) Total OXPHOS (Complex I–V) and UCP1 protein levels (expressed as % of WT) in isolated BAT mitochondria from chow-fed WT and adipocyte-specific SERCA2 KO mice. (K) OCR of the different mitochondrial complexes in isolated BAT mitochondria from chow-fed WT and adipocyte-specific SERCA2 KO mice. (L) OCR parameters in brown adipocytes differentiated from BAT SVF from chow-fed WT and adipocyte-specific SERCA2 KO mice. (M) BAT mitochondrial DNA (mtDNA) levels in chow-fed WT and adipocyte-specific SERCA2 KO mice, and (N) Mitotracker Green dye intensity in brown adipocytes differentiated from BAT SVF from chow-fed WT and adipocyte-specific SERCA2 KO mice. (O) Fgf21 expression in IWAT, BAT and liver in chow-fed WT and adipocyte-specific SERCA2 KO mice. (P) FGF21 protein levels in IWAT and BAT from chow-fed WT and adipocyte-specific SERCA2 KO mice. All values (N = 3–6) are expressed as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 5: Expression and secretion of FGF21 in mice after AMI. (A) Western blot analysis of hepatic FGF21 in WT or Liver SIRT5 OE 5 days after sham surgery or AMI. Expression levels of FGF21 were normalized to ß-tubulin (n = 3). *p < 0.05 comparing with sham surgery (n = 3). (B) Comparation of serum FGF21 level in mice before and after AMI (n = 5). **p < 0.01. (C) Western blot analysis of cardiac FGF21 in WT or Liver SIRT5 OE 5 days after sham surgery or AMI. Expression levels of FGF21 were normalized to ß-tubulin (n = 3). *p < 0.05.
Supplier Page from Abcam for Mouse FGF-21 ELISA Kit