Reducing VEGFB accelerates NAFLD and insulin resistance in mice via inhibiting AMPK signaling pathway

Figures

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  Fig. 1. The construction of VEGFB gene knockout mice. A The structure of VEGFB gene. B Process of constructing systemic of VEGFB gene knockout mice by Crispr/Cas9

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  Fig. 2. Appraisal of VEGFB gene knockout mice and NAFLD cell model with low expression of VEGFB. A VEGFB gene identification in wild-type and homozygous-type mice. B Relative mRNA expression level of VEGFB gene in heart and muscle tissue. C The content of VEGFB was detected by Elisa in the plasma of VEGFB systemic knockout wild-type and homozygous-type mice. D iWAT、eWAT and liver tissue of VEGFB gene knockout mice were analyzed by Western Blot. E VEGFB Protein expression level in iWAT、eWAT and liver tissue of VEGFB gene knockout mice. F–H Analyses of relative mRNA level of iWAT, eWAT and liver tissue in 3 groups of mice by RT-PCR. I HepG2 cell viability was induced by different concentrations of PA. J, K Levels of TG and TCHO in HepG2 induced by PA. L Western blot test of VEGFB in HepG2 cells after siRNA transfection. M Expression level of VEGFB protein in NAFLD cell model. N Relative mRNA expression level of VEGFB gene in NAFLD cell model. N = 6 cases per group. A–C * indicates vs. VEGFB+/+. D–H * indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05. I–N * indicates vs. NC, p < 0.05

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  Fig. 3. VEGFB gene knockout accelerated fat accumulation induced by HFD. A Monitoring of growth curve of mice aged 4–20 weeks. B Morphological pictures of iWAT, eWAT, liver and mice aged 20 weeks. C The relative weight of fat in 20-week-old mice. D The relative mass ratio of systemic fat, lean and liquid in 20-week-old mice. E HE staining pictures of iWAT and eWAT under a 400X light microscope. F, G Average area and average diameter of fat cells in iWAT and eWAT. H Weight of liver. N = 8 cases per group. * indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05

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  Fig. 4. VEGFB gene suppression induced disorder of lipid metabolism in NAFLD mice. A–D Changes of TG、TC、LDL and HDL of plasma and serum in 20-week-old mice. E–H Changes of TG、TC、LDL and HDL in HepG2 cells induced by PA solution. N = 8 cases per group. A-D * indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05. E–H * indicates vs. NC, p < 0.05

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  Fig. 5. Systemic knockout of VEGFB accelerates liver injury in NAFLD mice. A Morphological pictures of mice liver tissue stained by HE and Masson. a indicates macrovesicular steatosis, b indicates vesicular steatosis, c indicates hypertrophic hepatocytes, d indicates inflammatory cell aggregates. B–E Percentage of hepatic bullous steatosis, vesicular steatosis, hypertrophic area of hepatocytes and number of inflammatory cell aggregates in mice liver tissue. F Score of hepatic bullous steatosis, vesicular steatosis, hypertrophic area of hepatocytes and inflammatory cell aggregates and total score of NAFLD. G, H TG and TCHO levels in liver tissue. I, J AST and ALT levels in mice serum. N = 8 cases per group. *indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05

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  Fig. 6. VEGFB gene knockout accelerates insulin resistance in mice. A, B Curve monitoring of FBG and PBG in 4–20 week-old mice. C GTT. D Area of GTT under the curve. E ITT. F Area of ITT under the curve. G The relative value of blood glucose decrease. H Index of insulin resistance. I Insulin sensitivity index. N = 8 cases per group. J Serum insulin level after glucose stimulation for 30 min. *indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05

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  Fig. 7. The suppression of VEGFB gene inhibits AMPK phosphorylation level. A Venn diagram illustrates the number of loci with gained VEGFB and AMPK binding in liver. B Bioinformatics analysis of co-expressed genes of VEGFB and AMPK. C p-AMPK, AMPK, CaMKK2 in NAFLD mice model were analyzed by Western blot. D Protein expression level of p-AMPK/AMPK in NAFLD mice model. E Protein expression level of CaMKK2 in NAFLD mice model. F p-AMPK, AMPK, CaMKK2 in NAFLD cell model were analyzed by Western blot. G Protein expression level of p-AMPK/AMPK in NAFLD cell model. H Protein expression level of CaMKK2 in NAFLD cell model. N = 6 cases per group. D, E * indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05. G, H * indicates vs. NC, p < 0.05

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  Fig. 8. The suppression of VEGFB gene inhibits the AMPK/ACC/CPT1 signal pathway of fatty acid oxidation. A Western blot of p-ACC, ACC, CPT1 in NAFLD mice model were analyzed. B Protein level of p-ACC/ACC in NAFLD mice model. C Protein level of CPT1 in NAFLD mice model. D Western blot of p-ACC, ACC and CPT1 protein in NAFLD cell model were analyzed. E Protein level of p-ACC/ACC in NAFLD cell model. F Protein level of CPT-1 in NAFLD cell model. G mRNA level of CPT1α and Lcad in NAFLD mice model. H mRNA level of CPT1α and Lcad in NAFLD cell model. N = 6 cases per group. B, C, G *indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05; E, F, H * indicates vs. NC, p < 0.05

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  Fig. 9. The suppression of VEGFB gene stimulated the SREBP1/Scd1 signal pathway of lipid synthesis. A Western blot of SREBP1 and Scd1 in NAFLD mice model were analyzed. B Protein level of SREBP1 in NAFLD mice model. C Protein level of Scd1 in NAFLD mice model. D Western blot of SREBP1 and Scd1 in NAFLD cell model were analyzed. E Protein level of SREBP1 in NAFLD cell model. F Protein level of Scd1 in NAFLD cell model. G mRNA level of ACC1 and FAS in NAFLD mice model. H mRNA level of ACC1 and FAS in NAFLD cell model. N = 6 cases per group. B, C, G * indicates vs. SD, p < 0.05; # indicates vs. HFD, p < 0.05; E, F, H * indicates vs. NC, p < 0.05

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  Fig. 10. Pattern diagram of VEGFB gene participating in NAFLD through AMPK/ACC and AMPK/SREBP1 signal pathway