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APPL1 counteracts obesity-induced vascular insulin resistance and endothelial dysfunction by modulating the endothelial production of nitric oxide and endothelin-1 in mice.

Wang Y, Cheng KK, Lam KS, Wu D, Wang Y, Huang Y, Vanhoutte PM, Sweeney G, Li Y, Xu A - Diabetes (2011)

Bottom Line: However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear.In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Objective: Insulin stimulates both nitric oxide (NO)-dependent vasodilation and endothelin-1 (ET-1)-dependent vasoconstriction. However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear. This study aimed to investigate the roles of the multidomain adaptor protein APPL1 in modulating vascular actions of insulin in mice and in endothelial cells.

Research design and methods: Both APPL1 knockout mice and APPL1 transgenic mice were generated to evaluate APPL1's physiological roles in regulating vascular reactivity and insulin signaling in endothelial cells.

Results: Insulin potently induced NO-dependent relaxations in mesenteric arteries of 8-week-old mice, whereas this effect of insulin was progressively impaired with ageing or upon development of obesity induced by high-fat diet. Transgenic expression of APPL1 prevented age- and obesity-induced impairment in insulin-induced vasodilation and reversed obesity-induced augmentation in insulin-evoked ET-1-dependent vasoconstriction. By contrast, genetic disruption of APPL1 shifted the effects of insulin from vasodilation to vasoconstriction. At the molecular level, insulin-elicited activation of protein kinase B (Akt) and endothelial NO synthase and production of NO were enhanced in APPL1 transgenic mice but were abrogated in APPL1 knockout mice. Conversely, insulin-induced extracellular signal-related kinase (ERK)1/2 phosphorylation and ET-1 expression was augmented in APPL1 knockout mice but was diminished in APPL1 transgenic mice. In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.

Conclusions: APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

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Effects of siRNA-mediated knockdown of APPL1 on insulin-induced Akt signaling and NO production in HUVECs. Cells were transfected with siRNA specific for APPL1 (siAPPL1) or scrambled control for 40 h, followed by serum starvation for 6 h and stimulation with insulin (50 nmol/L) for 10 min. The expression of APPL1 (A), phosphorylation of Akt at Thr308 (B), and phosphorylation of eNOS at Ser1177 (C) were determined by Western blotting. NO release in the conditioned medium was measured at 60 min after insulin treatment. The data were expressed as the fold over the control cells treated without insulin (D). E: Cells were cotransfected with siAPPL1 or scrambled control plus a plasmid encoding HA-tagged Akt, followed by insulin treatment as above. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies and then probed with anti-Akt or anti-TRB3 antibody as indicated. T, total; p, phosphorylated. **P < 0.01 (n = 5–6).
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Figure 6: Effects of siRNA-mediated knockdown of APPL1 on insulin-induced Akt signaling and NO production in HUVECs. Cells were transfected with siRNA specific for APPL1 (siAPPL1) or scrambled control for 40 h, followed by serum starvation for 6 h and stimulation with insulin (50 nmol/L) for 10 min. The expression of APPL1 (A), phosphorylation of Akt at Thr308 (B), and phosphorylation of eNOS at Ser1177 (C) were determined by Western blotting. NO release in the conditioned medium was measured at 60 min after insulin treatment. The data were expressed as the fold over the control cells treated without insulin (D). E: Cells were cotransfected with siAPPL1 or scrambled control plus a plasmid encoding HA-tagged Akt, followed by insulin treatment as above. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies and then probed with anti-Akt or anti-TRB3 antibody as indicated. T, total; p, phosphorylated. **P < 0.01 (n = 5–6).

Mentions: Consistent with the above findings in APPL1 KO mice, insulin-induced phosphorylation of Akt at Thr308 and that of eNOS at Ser1177 was significantly decreased by siRNA-mediated knockdown of APPL1 in HUVECs (Fig. 6A–C). Insulin-induced elevation in endothelial NO production was also reduced by siRNA-mediated suppression of APPL1 expression (Fig. 6D). These changes were accompanied by an elevated interaction between Akt and its endogenous inhibitor Tribbles 3 (TRB3) (21), as determined by coimmunoprecipitation analysis (Fig. 6E).


APPL1 counteracts obesity-induced vascular insulin resistance and endothelial dysfunction by modulating the endothelial production of nitric oxide and endothelin-1 in mice.

Wang Y, Cheng KK, Lam KS, Wu D, Wang Y, Huang Y, Vanhoutte PM, Sweeney G, Li Y, Xu A - Diabetes (2011)

Effects of siRNA-mediated knockdown of APPL1 on insulin-induced Akt signaling and NO production in HUVECs. Cells were transfected with siRNA specific for APPL1 (siAPPL1) or scrambled control for 40 h, followed by serum starvation for 6 h and stimulation with insulin (50 nmol/L) for 10 min. The expression of APPL1 (A), phosphorylation of Akt at Thr308 (B), and phosphorylation of eNOS at Ser1177 (C) were determined by Western blotting. NO release in the conditioned medium was measured at 60 min after insulin treatment. The data were expressed as the fold over the control cells treated without insulin (D). E: Cells were cotransfected with siAPPL1 or scrambled control plus a plasmid encoding HA-tagged Akt, followed by insulin treatment as above. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies and then probed with anti-Akt or anti-TRB3 antibody as indicated. T, total; p, phosphorylated. **P < 0.01 (n = 5–6).
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Figure 6: Effects of siRNA-mediated knockdown of APPL1 on insulin-induced Akt signaling and NO production in HUVECs. Cells were transfected with siRNA specific for APPL1 (siAPPL1) or scrambled control for 40 h, followed by serum starvation for 6 h and stimulation with insulin (50 nmol/L) for 10 min. The expression of APPL1 (A), phosphorylation of Akt at Thr308 (B), and phosphorylation of eNOS at Ser1177 (C) were determined by Western blotting. NO release in the conditioned medium was measured at 60 min after insulin treatment. The data were expressed as the fold over the control cells treated without insulin (D). E: Cells were cotransfected with siAPPL1 or scrambled control plus a plasmid encoding HA-tagged Akt, followed by insulin treatment as above. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies and then probed with anti-Akt or anti-TRB3 antibody as indicated. T, total; p, phosphorylated. **P < 0.01 (n = 5–6).
Mentions: Consistent with the above findings in APPL1 KO mice, insulin-induced phosphorylation of Akt at Thr308 and that of eNOS at Ser1177 was significantly decreased by siRNA-mediated knockdown of APPL1 in HUVECs (Fig. 6A–C). Insulin-induced elevation in endothelial NO production was also reduced by siRNA-mediated suppression of APPL1 expression (Fig. 6D). These changes were accompanied by an elevated interaction between Akt and its endogenous inhibitor Tribbles 3 (TRB3) (21), as determined by coimmunoprecipitation analysis (Fig. 6E).

Bottom Line: However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear.In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Objective: Insulin stimulates both nitric oxide (NO)-dependent vasodilation and endothelin-1 (ET-1)-dependent vasoconstriction. However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear. This study aimed to investigate the roles of the multidomain adaptor protein APPL1 in modulating vascular actions of insulin in mice and in endothelial cells.

Research design and methods: Both APPL1 knockout mice and APPL1 transgenic mice were generated to evaluate APPL1's physiological roles in regulating vascular reactivity and insulin signaling in endothelial cells.

Results: Insulin potently induced NO-dependent relaxations in mesenteric arteries of 8-week-old mice, whereas this effect of insulin was progressively impaired with ageing or upon development of obesity induced by high-fat diet. Transgenic expression of APPL1 prevented age- and obesity-induced impairment in insulin-induced vasodilation and reversed obesity-induced augmentation in insulin-evoked ET-1-dependent vasoconstriction. By contrast, genetic disruption of APPL1 shifted the effects of insulin from vasodilation to vasoconstriction. At the molecular level, insulin-elicited activation of protein kinase B (Akt) and endothelial NO synthase and production of NO were enhanced in APPL1 transgenic mice but were abrogated in APPL1 knockout mice. Conversely, insulin-induced extracellular signal-related kinase (ERK)1/2 phosphorylation and ET-1 expression was augmented in APPL1 knockout mice but was diminished in APPL1 transgenic mice. In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.

Conclusions: APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

Show MeSH
Related in: MedlinePlus