The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium
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Figures
- FIG. 1.
Haploinsufficiency of IGF-1R at a whole-body level leads to resistance to IGF-1–mediated glucose lowering and glucose intolerance but enhanced insulin sensitivity. A: Intraperitoneal glucose tolerance test. B: Intraperitoneal insulin tolerance test. C: Intraperitoneal IGF-1 tolerance test. Fasting plasma insulin (D) and fasting plasma IGF-1 (E) in mice with haploinsufficiency of IGF-1R and wild-type littermates. *P < 0.05; at least n = 10 per group for all experiments. Data shown as mean ± SEM.
- FIG. 2.
Haploinsufficiency of IGF-1R at a whole-body level leads to resistance to IGF-1–mediated aortic relaxation, blunted constriction to PE, and enhanced basal aortic NO production. Endothelial function in aortic rings from mice with haploinsufficiency of IGF-1R and wild-type littermates. A: Vasorelaxation in response to IGF-1 in wild type. B: IGF-1 relaxation is blunted in IGF-1R–deficient mice. C: eNOS activity in response to IGF-1 is blunted in IGF-1R haploinsufficient mice. D and E: Constriction in response to l-NMMA in wild-type mice (D) and IGF-1R haploinsufficient mice (E). F: Maximal constriction (Emax) in response to l-NMMA indicative of NO bioavailability is significantly higher in IGF-1R haploinsufficient mice. G: Constriction dose–response curve to PE in wild-type and IGF-1R haploinsufficient mice. H: Maximal constriction in response to PE is reduced in mice with haploinsufficiency of IGF-1R. I: IGF-1R expression in isolated pulmonary ECs in mice with haploinsufficiency of IGF-1R and wild-type littermates. *P < 0.05; at least n = 10 per group for all experiments.
- FIG. 3.
Haploinsufficiency of IGF-1R at a whole-body level leads to increased levels of tyrosine-phosphorylated IR, increased insulin-mediated NO production, and serine phosphorylation of eNOS in ECs. A: Enhanced basal tyrosine phosphorylation of IR in isolated ECs from IGF-1R haploinsufficient mice. B: Increased insulin-mediated NO production in ECs from IGF-1R haploinsufficient mice (bottom panel shows representative time series graph for change of DAF-FM fluorescence in pulmonary ECs in response to insulin [100 nmol/L]). C: Serine phosphorylation of eNOS is enhanced in response to insulin in ECs from IGF-1R haploinsufficient mice. D: No difference in IR levels in ECs from IGF-1R haploinsufficient mice. E: Reduced IGF-1R levels in ECs from IGF-1R haploinsufficient mice. F: Reduced hybrid receptors in ECs from IGF-1R haploinsufficient mice. *P < 0.05; n = 5–10 mice per experiment. AU, arbitrary unit; IB, immunoblotted; WT, wild type. (A high-quality color representation of this figure is available in the online issue.)
- FIG. 4.
Metabolic profiles of mice with haploinsufficiency and holoinsufficiency of IGF-1R in the endothelium. A and B: Glucose tolerance tests (A) and insulin tolerance tests (B) are similar in wild type and mice with haploinsufficiency of IGF-1R in the endothelium. C and D: Glucose tolerance tests (C) and insulin tolerance tests (D) are similar in wild type and mice with holoinsufficiency of IGF-1R in the endothelium. E and F: Fasting blood glucose (E) and fasting insulin levels (F) are similar in wild-type mice and mice with haploinsufficiency and holoinsufficiency of IGF-1R in the endothelium. At least n = 4 mice per group for all experiments. (A high-quality color representation of this figure is available in the online issue.)
- FIG. 5.
Vascular responses in aortic rings of mice with haploinsufficiency and holoinsufficiency of IGF-1R in the endothelium. A: Constrictor responses to PE in rings preincubated with l-NMMA in wild-type mice. B: Enhanced constrictor responses to PE in mice with EC-specific haploinsufficiency of IGF-1R. C: Constrictor responses to PE in rings preincubated with l-NMMA in wild-type mice. D: Enhanced constrictor responses (indicative of increased NO bioavailability) in rings in mice with EC-specific holoinsufficiency of IGF-1R. E: Blunted constrictor responses to PE in mice with EC-specific haploinsufficiency of IGF-1R. F: Blunted constrictor responses to PE in mice with EC-specific holoinsufficiency of IGF-1R. G: Maximal change in tension in response to l-NMMA in wild-type mice, mice with EC-specific haploinsufficiency of IGF-1R, and mice with EC-specific holoinsufficiency of IGF-1R, demonstrating gene dose effect of IGF-1R on NO bioavailability. H: Maximal change in tension in response to PE in wild-type mice, mice with EC-specific haploinsufficiency of IGF-1R, and mice with EC-specific holoinsufficiency of IGF-1R, demonstrating gene dose effect of IGF-1R on constrictor responses to PE. I: Quantification of hybrid receptors in wild-type mice, mice with EC-specific haploinsufficiency of IGF-1R, and mice with EC-specific holoinsufficiency of IGF-1R. All experiments had at least n = 5 mice. *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)
- FIG. 6.
Deletion of IGF-1R in mice with haploinsufficiency of IR (IR+/−) restores insulin-stimulated NO production. A: Glucose tolerance tests in IR+/− and IR+/−/IGF-1R+/− mice. B: Insulin tolerance tests in IR+/− and IR+/−/IGF-1R+/− mice. C: Deletion of IGF-1R in IR+/− mice restores insulin-mediated eNOS activation. D: Insulin-mediated aortic relaxation is blunted in IR+/− mice. E and F: Deletion of IGF-1R in IR+/− mice restores insulin-mediated relaxation in aortic rings (E) and maximal relaxation to insulin in IR+/− and IR+/−/IGF-1R+/− mice (F). G: Insulin-mediated NO release in response to insulin in isolated ECs of IR+/− mice is restored by deletion of IGF-1R. H: Representative time series graph for change of DAF-FM fluorescence in pulmonary ECs in response to insulin. I: Hybrid receptor expression (100 nmol/L). All experiments had at least n = 6 animals. *P < 0.05. AU, arbitrary unit; IB, immunoblotted; IP, immunoprecipitation. (A high-quality color representation of this figure is available in the online issue.)
- FIG. 7.
siRNA-mediated knockdown of IGF-1R in HUVECs reproduces the in vivo scenario, increasing EC insulin-mediated eNOS activation. A: Relative levels of IGF-1R measured using quantitative PCR in siRNA and scrambled peptide–treated HUVECs. B: No difference in IR RNA. C: PCR gel showing specific effect of siRNA on IR and IGF-1R in HUVEC. D: Reduced hybrid receptors in siRNA-treated HUVECs. E: Increased basal and insulin-mediated eNOS serine phosphorylation in siRNA-treated cells. F: Increased basal and insulin-mediated eNOS activation in siRNA-treated cells. All data are mean of at least three experiments. *P < 0.05; #P < 0.05 vs. control. AU, arbitrary unit; Con, Ctrl, control; Scr, Scram, scrambled.
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