Pulmonary vascular effect of insulin in a rodent model of pulmonary arterial hypertension

Fig. 1.. Induced hyperglycemia and hyperinsulinemia models in control and BMPR2-mutant mice. (a, b) STZ and WD resulted in hyperglycemia with insulin levels being elevated most in animals fed WD. (c, d) Markers of insulin resistance were elevated by WD feeding. Comparison with SD-fed group of same genotype, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 7–10 per bar.

Fig. 2.. Western diet raises RVSP in both control animals and a transgenic mouse model of human PAH. (a) RVSP was elevated by WD regardless of genotype. Treatment with STZ had no effect on RVSP. (b) RV BNP expression was affected by diabetogenic exposure (two-way ANOVA P < 0.05), without genotype effect. (c) Fulton index was no different across groups. (d) Reduced LVEF did not explain WD-induced PH. (e) Cardiac output did not vary by genotype or diet. Symbols are same as in Fig. 1. n = 3–4 per bar for BNP expression, otherwise 5–12 per bar.

Fig. 3.. Hyperinsulinemia, but not hyperglycemia, is associated with RVSP in control and BMPR2 mutant mice. Pearson correlations are shown for fasting insulin (a, b), body weight (c, d), and fasting glucose (e, f). There was strong correlation of RVSP with both weight and insulin. n = 15–21 for each correlation. Symbols denoting statistical significance of the R2 value are the same as given in Fig. 1.

Fig. 4.. BMPR2 mutation and WD have varied results on small pulmonary vessels. (a) Small pulmonary vessel density was not altered by diet or genotype. Mean vessels per 10× high power field is given (averaged over ten fields, n = 3–5 mice per bar). (b) Pulmonary vessels in mice with BMPR2 mutation showed increased medial wall thickness, but this did not vary based on diet exposure (two-way ANOVA P < 0.01 for effect of genotype, P = NS for effect of diabetogenic exposure). The mean medial wall thickness from all vessels on 15 high powered fields is given for n = 4 mice. (c) WD and mutation each significantly increased lung ROS (P < 0.01 for WD, P < 0.05 for BMPR2 mutation, n = 10–12 per bar). ROS given as fold change compared with control genotype, SD group. Comparison with SD-fed group of same genotype, *P < 0.05.

Fig. 5.. BMPR2 mutation increases insulin signaling protein expression in lung. Total and phosphorylated Akt (a–c) and AMPK (d–f) were increased by the presence of BMPR2 mutation compared to control. WD increased phosphorylation of Akt and AMPK in control mice, but in BMPR2 mutant animals no further increase was seen compared with SD fed animals. Comparison with SD-fed group of same genotype, *P < 0.05, ***P < 0.001. BMPR2R899X animals compared with littermate control, †P < 0.05, ††P < 0.01, †††P < 0.001, ††††P < 0.0001. ‡Effect of mutation P < 0.05 by two-way ANOVA. n = 3 mice per bar run in technical triplicate.

Fig. 6.. Insulin-induced glucose flux is reduced by BPMR2 mutation in isolated endothelial cells. (a) The rate of utilization of glucose from supernatant media was reduced by BMPR2 mutation with Mutation1 having a more profound effect than Mutation2. (b) There was increased intracellular glucose analogue in PMVECs under insulin stimulation, an effect significantly reduced by the presence of BMRP2 mutation. *P < 0.05 compared with cells without BMPR2 mutation. Six technical replicates used for each experiment.

Fig. 7.. Expression of insulin receptor isoforms and downstream insulin signaling molecules in cultured endothelial cells with and without BMPR2 mutation. (a) Insulin receptor isoform A was not differentially expressed according to mutation status. Isoform B expression varied by mutation status, but the ratio was not skewed significantly towards increased presence of IR-A. (b) The MAPK signaling molecule Erk1/2 had no statistically significant change in phosphorylation by mutation or insulin status. (c–e) Akt, however, had reduced expression with one of two mutations in BMPR2 but relative phosphorylation of the Ser473 site remained intact. *P < 0.05 compared with cells without mutation (two-way ANOVA); †P < 0.05 compared with Mutation1 (two-way ANOVA); ‡P < 0.05 compared with no insulin (two-way ANOVA).

Fig. 8.. Insulin-induced translocation of glucose transporter type 4 is reduced by BMPR2 mutation. GLUT4 immunolocalization (green) was demonstrated at various time points of insulin exposure in endothelial cells co-stained with DAPI (blue) and actin (red). The proportion of cells with GLUT4 predominantly located in a perinuclear region versus diffusely throughout the cytoplasm was represented graphically. *P < 0.05 compared to basal condition (zero minutes); ‡P < 0.05 compared to cells without mutation. Statistical comparison is by chi-square test.