Effects of imatinib on vascular insulin sensitivity and free fatty acid transport in early weight gain

Figures

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  Fig 1. The effect of imatinib and Western Diet (WD) on body weight, metabolic insulin sensitivity and glucose metabolism. A: WD-fed mice had higher body weight than chow fed mice after two weeks of diet (P = 0.007, n = 8). B: Two-week WD feeding caused bodyweight gain in mice given saline injections. This weight-gaining effect was rescued by imatinib treatment (P = 0.006, n = 8). C: Glucose infusion rate (GIR) quantifies whole-body glucose uptake during the hyperinsulinemic-euglycemic clamp. Two-week Western diet feeding and imatinib treatment did not affect GIR: P = 0.42 and P = 0.32, respectively (n = 5–7). D: Two-week Western diet feeding and imatinib treatment did not affect fasting glucose levels: P = 0.07 and P = 0.23, respectively (n = 7–8). Results were tested for significance with two-way ANOVA tests. **P<0.01.

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  Fig 2. Vasoreactivity of the gracilis artery in response to insulin, measured with intravital microscopy. A: Imatinib does not affect insulin-induced vasoreactivity in vivo in mice fed a chow diet. B: Imatinib increases insulin-induced diameter change at t = 60 minutes in mice fed a Western diet (WD). *P<0.05 as tested with unpaired T-test; n = 4–8 per group.

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  Fig 3. Effect of WD and imatinib on triglyceride (TG) content of skeletal muscle, heart, and plasma and on muscle gene expression. A: TG content in skeletal muscle: WD increased TG content in skeletal muscle as compared to chow (P = 0.009). Imatinib did not decrease TG content in the chow group (P>0.9) and in the WD group (P = 0.5). B: TG content in heart: WD caused no increase in TG content as compared to chow diet (P = 0.1). Imatinib caused no effect on TG content. C: TG content in plasma: imatinib did not decrease the TG content in the plasma of WD fed mice. D: Expression of VEGF-B in gastrocnemeus muscle: imatinib (P = 0.27) and WD (P = 0.7) did not affect VEGF-B expression. E: Expression of VEGFR1 in gastrocnemeus muscle: WD feeding increased VEGFR1 expression compared to Chow feeding (P = 0.04), while imatinib did not affect the expression (P = 0.5). F: Expression of IRS1 in gastrocnemeus muscle: imatinib (P = 0.08) and WD (P = 0.7) did not affect IRS1 expression. G: Expression of Glut4 in gastrocnemeus muscle: WD feeding increased Glut4 expression compared to Chow feeding (P = 0.04), imatinib had no effect on the expression (P = 0.5). *P<0.05 and **P<0.01 in Bonferroni post hoc…

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  Fig 4. Effect of vascular endothelial growth factor-B (VEGF-B) and imatinib on Crk-like protein (CrkL) phosphorylation. A: Western blot images of VEGF-B-mediated phosphorylation of CrkL at different time points. C: Quantification of A. The phosphorylation of CrkL showed an increase at 15 min stimulation with VEGF-B. *P = 0.019 in Bonferroni post hoc test of repeated measures one-way ANOVA. B: Western blot images of the effect of imatinib on VEGF-B-mediated CrkL phosphorylation. D: Quantification of B. Imatinib attenuates the phosphorylation of CrkL in conditions with 15 and 30 min stimulation with VEGF-B. **P<0.01, *P<0.05 in Bonferroni post hoc test of repeated measures two-way ANOVA. a.u. = arbitrary units; n = 3. For uncropped blot images see S1 Fig.

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  Fig 5. Effect of vascular endothelial growth factor-B (VEGF-B) and imatinib on ERK1/2 phosphorylation. A: Western blot images of VEGF-B mediated phosphorylation of ERK1/2 at different time points. C: Quantification of A. The treatment of HUVECS with VEGF-B enhanced the phosphorylation of ERK1/2 at 15 min stimulation with VEGF-B. *P = 0.04 in Bonferroni post hoc test of repeated measures one-way ANOVA. B: Western blot images of the effect of imatinib on VEGF-B mediated ERK1/2 phosphorylation (“-” = DMSO; “+” = Imatinib). D: Quantification of B. Imatinib caused no effect on the phosphorylation of ERK1/2 in basal conditions and in conditions treated with VEGF-B. *P<0.05 in Bonferroni post hoc test of repeated measures two-way ANOVA. a.u. = arbitrary units; n = 3. For uncropped blot images see S1 Fig.

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  Fig 6. Effect of VEGF-B and imatinib on Bodipy-FA uptake into HUVECS. A: Bodipy-FA uptake in cells treated with VEGF-B 100 ng/ml and VEGF-B 300 ng/ml at different time points. B: Quantification of fluorescence in A. VEGF-B 100 ng/ml and 300 ng/ml caused a time-dependent increase in the uptake of Bodipy-FA at 2 hours (P = 0.02 and P = 0.02) and 6 hours (P = 0.01 and P = 0.03) of VEGF-B stimulation. *P in ordinary two-way ANOVA. a.u. = arbitrary units; n = 6. The data in this experiment are partly the same as the data in experiment Fig C, D. C: BODIPY-FA uptake into HUVECS treated with imatinib, with or without 6h 100 ng/ml VEGF-B stimulation. D: Quantification of fluorescence in B. Six hours stimulation with 100ng/ml VEGF-B caused an increase in the uptake of fatty acids in HUVECS. *P = 0.02 in ordinary one-way ANOVA. However, imatinib caused no decrease in Bodipy-FA uptake under basal and VEGF-B treated conditions. *P = 0.96 and P = 0.32 in ordinary one-way ANOVA. Non-significant (ns). N = 5–14.